JP5830460B2 - Compounds, compositions and methods for treating cancer and fibrotic diseases - Google Patents

Description

  This application claims priority from US Provisional Patent Application No. 61/162348, filed March 23, 2009, which is incorporated herein by reference in its entirety.

The present invention relates to a molecule targeting and internalized by an ENDO180 polypeptide, a conjugate comprising the molecule, a composition comprising the molecule and the conjugate, a cell proliferative disease or disorder, and fibrosis As well as methods of using them to deliver therapeutic agents to cells expressing an ENDO180 polypeptide on the surface of the cells, as well as to control (regulate) tumor progression.

ENDO180 , also known as ENDO180 receptor CD280, uPARAP (urokinase-type plasminogen activator receptor-related protein), and mannose receptor type C2 (MRC2), is recycled, inducing binding ligands to degradation in endosomes. It is an endocytic receptor. It is involved in the production of plasmin from plasminogen because it is part of a triple complex with urokinase-type plasmin activator (uPA) and urokinase-type plasmin activator receptor (uPAR). Plasmin has also been shown to play a role in both extracellular matrix (ECM) turnover and proteolytic conversion of latent TGF-β to the active form.

  In addition to its role in plasmin production, the ternary complex is involved in the activation of matrix metalloproteinase (MMP) proenzymes that act on fibrin and bind to several collagens, and the general turnover of the extracellular matrix It became clear to do. This complex is also involved in cell adhesion and signal transduction (Bherendel al, 2000. JBC 275: 1993-2002).

ENDO180 is a recycled endocytosis receptor that functions in cell motility and extracellular matrix remodeling by promoting cell migration and collagen uptake for intracellular degradation (Niels. 2004). Biol Chem. 385 (2): 103-36; Kjoller et al, 2004 Exp Cell Res. 293 (1): 106-16; Wienke et al., 2007 Cancer Res. 67 (21): 10230-40). ENDO180 is mannose receptor is a macrophage mannose receptor family, phospholipase _{A 2,} and the DEC-205 / MR6 shares homology (Isacke et al, 1990 Mol Cell.Biol.10 :.. 2606-2618; Sheikh et al., 2000, J. Cell. Sci. 113: 1021-1032; Behrendt et al., 2000, J. Biol. Chem. 275: 1993-2002). This family class is divided into large extracellular domains, including cysteine-rich domains, fibronectin type II domains (FNII), and N-terminal signal sequences followed by 8 or 10 C-type lectin-like domains (CTLDs), and It is based on conservation of the overall structure, such as a small transmembrane domain and an intracellular domain (total of about 66 amino acids). As a family, these receptors have two prominent features: First, they belong to a large C-type lectin superfamily, but have multiple CTLDs within a single polypeptide backbone. Contains independently (Taylor ME, 1997 Glycobiology 7: v-vii, McKay et al, 1998, Eur. J. Immunol. 28: 4071-4083, Howard and Isacke, 2002, supra). Second, they share the ability to be recycled between the plasma membrane of the cell and the intercellular compartment (Isacke et al, 1990, supra, Zvaritch et al., 1996, J. Biol. Chem. 271: 250-257). ENDO180 is unique among the mannose receptor family in that it is targeted from the plasma membrane to the recycle endosome rather than to the late endosome / lysosome compartment (Howard and Isacke, 2002, supra).

  ENDO180 is a clathrin-covered pit on the cell surface (Isacke et al., 1990 Mol. Cell. Biol. 10: 2606-2618; Sheikh et al., 2000, J. Cell. Sci. 113: 1021- 1032), and is localized in endosomes. It is mainly expressed in fibroblasts, endothelial cells, and macrophages. In situ hybridization showed its expression in highly vascularized organs. ENDO 180 is also the osteogenic region of mouse embryos (Wu et al., 1996, J. Biol. Chem. 271: 21323-21330), and the osteoblasts at sites of developing endochondral and intramedullary ossification. It has also been found in cells and bone cells (Engelholm et al., 2001, Trends Cardiovasc. Med. 11: 7-13).

The following patent publications also relate to the ENDO180 receptor: US Pat. No. 6,117,977, US Pat. No. 7,399,468, WO 97/40154, and WO 00/58473. PCT Patent Application Publication No. WO 2004/100759 and US Patent Application Publication Nos. 2007/0072244 and 2009/0202566, which are hereby incorporated by reference in their entirety, to the applicant of the present invention are: It relates to a method for identifying compounds capable of modulating the activity of the human ENDO180 receptor.

Antibody therapy The search for new therapies to treat cancer and other diseases has led to the development of human and humanized antibodies capable of inhibiting receptor function. International Patent Application Publication No. WO 2006/023491 provides a method of RNA interference comprising contacting a cell with a fusion protein-double stranded RNA complex, the complex comprising a double stranded RNA of interest. And a fusion protein that is an antibody Fab fragment-protamine fusion protein.

  The present invention is based in part on the identification of isolated molecules that specifically bind to the ENDO180 polypeptide on the cell surface. In some embodiments, the molecule binds to the extracellular domain of the ENDO180 polypeptide and is internalized into the cell by the polypeptide, thereby causing the therapeutic and diagnostic cargo of the cell to express the ENDO180 polypeptide. Provide vehicles useful for delivery. Accordingly, in some embodiments, the present invention provides a conjugate comprising a molecule that specifically binds to an ENDO180 polypeptide and a therapeutic agent useful for delivery of the therapeutic agent into a cell. In some embodiments, the ENDO180 polypeptide is substantially identical to the amino acid sequence set forth in SEQ ID NO: 2 encoded by a polynucleotide that is substantially identical to the nucleic acid sequence set forth in SEQ ID NO: 1.

  In one aspect, the present invention provides an anti-ENDO180 antibody produced by a hybridoma cell line designated E3-8D8 (BCCM accession number LMBP 7203CB), or a fragment of said antibody, that binds to the ENDO180 receptor on the cell surface. provide. In some embodiments, binding of the antibody to the receptor results in internalization of the antibody into the cell. Also provided is an E3-8D8 hybridoma cell line.

  In some embodiments, the antibodies and fragments thereof are humanized or chimeric antibodies, or fragments thereof.

  The present invention provides a composition comprising at least one anti-ENDO180 antibody or fragment thereof produced by an E3-8D8 hybridoma or a humanized molecule, chimeric antibody or fragment thereof, and a carrier.

  In some embodiments, the isolated antibody is a full-length IgG, Fab fragment, Fab ′ fragment, F (ab ′) 2 fragment, their heavy and / or light chain variable portion, Fab miniantibody, And selected from the group consisting of scFv. In some embodiments, the antibody is a recombinant polypeptide comprising a heavy chain CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 7 or a variant thereof that retains the ability to specifically bind to ENDO180. In some embodiments, the antibody further comprises a light chain CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof, that retains the ability to specifically bind to ENDO180.

  In some embodiments, the antibody is an scFv recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof that retains the ability to specifically bind to ENDO180. In certain embodiments, an antibody that exhibits binding affinity for the ENDO180 receptor and comprises a CDR3 domain as set forth in SEQ ID NOs: 7 and 8 is directed to cells that express ENDO180 by the receptor when the antibody contacts the receptor. Internalized.

  The present invention further provides a composition comprising at least one anti-ENDO180 antibody or fragment thereof as described above and a moiety comprising a radioisotope, therapeutic agent, cytotoxic agent, or detectable label. In some embodiments, the moiety is attached to the antibody covalently by a linker or chemical bond, or non-covalently by an ionic bond, van der Waals bond, electrostatic bond, or hydrogen bond. (Or linked or conjugated).

In some embodiments,
a) a monoclonal antibody produced by the hybridoma cell line E3-8D8 (BCCM accession number LMBP 7203CB),
b) an antibody or fragment thereof that binds to the same epitope as the antibody of (a),
c) the humanized antibody of (a) or (b),
d) a fragment of an antibody comprising a polypeptide substantially similar to SEQ ID NO: 6, and e) a recombination comprising CDR3 having an amino acid sequence substantially similar to the amino acid sequences set forth in SEQ ID NOs: 7 and 8 Polypeptide,
There is provided an anti-ENDO180 antibody or antigen-binding fragment thereof selected from

further,
a) a monoclonal antibody produced by the hybridoma cell line E3-8D8 (BCCM accession number LMBP 7203CB),
b) an antibody or fragment thereof that binds to the same epitope as the antibody of (a),
c) the humanized antibody of (a) or (b),
d) a fragment of an antibody comprising a polypeptide substantially similar to SEQ ID NO: 6, and e) a recombination comprising CDR3 having an amino acid sequence substantially similar to the amino acid sequences set forth in SEQ ID NOs: 7 and 8 Polypeptide,
A composition comprising an anti-ENDO180 antibody or antigen-binding fragment thereof selected from

  In some embodiments, the composition further comprises a moiety that includes a radioisotope, a therapeutic agent, a cytotoxic agent, or a detectable label.

The present invention also provides
a) a monoclonal antibody produced by the hybridoma cell line E3-8D8 (BCCM accession number LMBP 7203CB),
b) an antibody or fragment thereof that binds to the same epitope as the antibody of (a),
c) the humanized antibody of (a) or (b),
d) a fragment of an antibody comprising a polypeptide substantially similar to SEQ ID NO: 6, and e) a recombinant comprising a CDR having an amino acid sequence substantially similar to the amino acid sequences set forth in SEQ ID NOs: 7 and 8. Polypeptide,
There is also provided a method of treating a subject suffering from a proliferative disease comprising administering to the subject a composition comprising an anti-ENDO180 antibody or antigen-binding fragment thereof selected from

  In some embodiments, the proliferative disease is selected from solid tumors, hematopoietic tumors, tumor metastases, fibrosis, and macrophage related diseases.

  In some embodiments, the tumor is an ovarian tumor, a breast tumor, an osteoblastic / osseous cell cancer, a prostate cancer, a head and neck cancer, a leukemia, a renal cell cancer, or a transitional cell carcinoma.

  In some embodiments, the fibrosis is liver fibrosis, myelofibrosis, renal fibrosis for any reason (CKD including end stage renal disease (ESRD)), pulmonary fibrosis (interstitial pulmonary fibrosis (ILF) )), All possible types of incidental and iatrogenic (surgical) skin damage and associated abnormal scars (keloids), scleroderma, cardiac fibrosis, glaucoma filtration surgery failure, intestinal adhesions .

  In some embodiments, the macrophage-related disease is inflammation or atherosclerosis.

In one aspect, the present invention provides
a) an antibody or antigen-binding portion thereof that specifically binds to the extracellular domain of the ENDO180 polypeptide on the cell surface;
b) a moiety containing a radioisotope, therapeutic agent, cytotoxic agent, or detectable label;
c) optionally, a linking moiety linking a) to b);
A conjugate is provided comprising:

  In some embodiments, the moiety is a therapeutic agent selected from oligonucleotide agents and non-oligonucleotide agents. In some embodiments, the therapeutic agent is an oligonucleotide therapeutic comprising an inhibitory oligonucleotide. Thus, in various embodiments, the therapeutic agent is selected from an antisense compound, a chemically modified siRNA compound, an unmodified siRNA compound, a chemically modified shRNA compound, an unmodified shRNA compound, a chemically modified miRNA compound, and an unmodified miRNA compound. . In various preferred embodiments, the therapeutic agent is a chemically modified siRNA. In some embodiments, the chemically modified siRNA compound inhibits expression of a target gene associated with cancer, fibrosis, or a macrophage-related disease. In some embodiments, the target gene is selected from any one of the target genes listed in Table A below.

  In certain embodiments, the therapeutic agent is attached to the antibody via a nucleotide linking moiety or a non-nucleotide linking moiety.

  In yet another aspect, the present invention provides a pharmaceutical composition comprising a conjugate of the invention.

  In yet another aspect, the invention comprises administering to a subject a therapeutically effective amount of an antibody that specifically binds to and is internalized by the ENDO180 polypeptide, wherein the antibody is shared Provided are methods of treating a subject suffering from a proliferative disease that is conjugated or non-covalently associated with a therapeutic agent.

In some embodiments, the proliferative disease is selected from malignant and benign proliferative diseases. In some embodiments, the proliferative disease is cancer. In other embodiments, the proliferative disorder is fibrosis. Non-limiting examples of diseases and disorders for use of the present invention include:
1. Soft tissue sarcoma (macrophage-monocyte activated myofibroblasts, neovascular cells, and infiltrating cells) in which ENDO180 is expressed in tumors and tumor stromal cells,
2. Cell carcinoma in which ENDO180 is expressed in tumor stromal cells (macrophage-monocyte activated myofibroblasts, neovascular cells, and infiltrating cells)
3. 3. Cell carcinoma that expresses ENDO180 and has acquired high metastatic potential due to epithelial-mesenchymal transition. For example, leukemia expressing ENDO180 from the macrophage-monocyte system. For example, renal, lung, and liver fibrotic diseases with activated myofibroblasts. Diseases and disorders associated with macrophages, including atherosclerosis and chronic inflammation.

The polynucleotide and amino acid sequences of various compounds according to the present invention are provided. Human ENDO180 mRNA (SEQ ID NO: 1) Human ENDO180 polypeptide (SEQ ID NO: 2) SEQ ID NO: 3 polynucleotide sequence of the extracellular domain of human ENDO180 (amino acids 1 to 522) having the FLAG sequence (FLAG domain is underlined) (pcDNA3-5'hendo180-FLAG construct, SEQ ID NO: 3) The polypeptide sequence of SEQ ID NO: 3 (SEQ ID NO: 4) polynucleotide sequence of scFv clone G7V (SEQ ID NO: 5) scFv clone G7V polypeptide G7V heavy chain CDR3 (SEQ ID NO: 7) sequence (SEQ ID NO: 6) G7V light chain CDR3 (SEQ ID NO: 8) Polypeptides 1 to 522 of the extracellular domain of human ENDO180 Internalization of CypHer5E fluorophore labeled anti-ENDO180 mAb into cells expressing ENDO180. Same as above Same as above Same as above Same as above Same as above Same as above Same as above Internalization of biotin-labeled anti-ENDO180 mAb into mice with unilateral ureter-occlusive kidneys. Internalization of anti-ENDO180 mAb conjugated to CypHer5E fluorophore into myelomonocytic human leukemia MonoMac cell line expressing ENDO180. Internalization of anti-ENDO180 mAb conjugated to CypHer5E fluorophore into myelomonocytic human leukemia MonoMac cell line expressing ENDO180.

Definitions For convenience, certain terms used in the specification, examples, and claims are set forth herein.

  It should be noted that as used herein, the singular forms “a”, “an”, and “the” include the plural unless the context clearly dictates otherwise.

  Where aspects or embodiments of the invention are described with respect to a Markush group or other alternative group, one of ordinary skill in the art will be able to describe the invention thereby with respect to any individual element or subgroup of elements of a group. You will recognize that.

  An “inhibitor” can reduce (partially or fully) the expression of a gene or the activity of the product of such a gene to a degree sufficient to achieve the desired biological or physiological effect. Compound. The term “inhibitor” as used herein includes one or more of oligonucleotide inhibitors, including siRNA, shRNA, synthetic shRNA, miRNA, antisense RNA and DNA, and ribozymes. “Inhibitory oligonucleotides” include antisense compounds, chemically modified siRNA compounds, unmodified siRNA compounds, chemically modified shRNA compounds, unmodified shRNA compounds, chemically modified miRNA compounds, and unmodified miRNA compounds.

  An “siRNA inhibitor” is a compound capable of reducing the expression of a gene or the activity of the product of such a gene to an extent sufficient to achieve the desired biological or physiological effect. As used herein, the term “siRNA inhibitor” refers to one or more of siRNA, shRNA, synthetic shRNA, and miRNA. Inhibition may be referred to as downregulation, or silencing for RNAi.

  As used herein, the term “inhibit” refers to reducing the expression of a gene or the activity of the product of such a gene to a degree sufficient to achieve the desired biological or physiological effect. Point to. Inhibition may be complete or partial. As used herein, the term “ENDO180 gene” preferably has 90% homology, more preferably 95% homology, even more preferably 98%, to the amino acid coding region of SEQ ID NO: 1. Defined as nucleic acid sequences that bind to the ENDO180 gene under highly stringent hybridization conditions, which are well known in the art (eg, Ausubel). et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1988), updated in 1995 and 1998).

  As used herein, the term “ENDO180” or “ENDO180 polypeptide” or “ENDO180 receptor” preferably has at least 90% homology, more preferably at least 95% homology to SEQ ID NO: 2. And even more preferably at least 98% homology, or any homologue of an ENDO180 polypeptide having 100% identity, either as its full length or fragment or domain, spliced variant nucleic acid sequence Mutants or polypeptides encoded by are defined as chimeras with other polypeptides, provided that any of the above has the same or substantially the same biological function as the ENDO180 receptor It shall have. ENDO180 polypeptide or ENDO180 polypeptide homologue represents a soluble protein, membrane-bound (either purified membrane preparation or on the cell surface), bead-bound, or ENDO180 protein or fragments and polypeptides derived therefrom. It may exist in different forms, including but not limited to any other form. The term “inhibition” as used herein refers to reducing the expression of a gene or the activity of the product of such a gene to an extent sufficient to achieve the desired biological or physiological effect. . Inhibition is either complete or partial.

  The terms “mRNA polynucleotide sequence”, “mRNA sequence”, and “mRNA” are used interchangeably.

  As used herein, the terms “polynucleotide” and “nucleic acid” may be used interchangeably and refer to a nucleotide sequence comprising deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is to be understood that the term includes equivalents of either RNA or DNA analogs made from nucleotide analogs. Throughout this application, mRNA sequences are described as representing the corresponding gene.

  “Oligonucleotide” or “oligomer” refers to a deoxyribonucleotide or ribonucleotide sequence of about 2 to about 50 nucleotides. Each DNA or RNA nucleotide may independently be natural or synthetic, and / or modified or unmodified. Modifications include changes to internucleotide linkages in sugar moieties, base moieties, and / or oligonucleotides. The compounds of the present invention include molecules comprising deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, and combinations thereof.

  Substantially complementary refers to greater than about 84% complementarity to another sequence. For example, in an overlapping region of 19 base pairs, one mismatch results in 94.7% complementarity, two mismatches yield approximately 89.5% complementarity, and three mismatches represent approximately 84.2% complementarity. Bring homology and make the overlapping region substantially complementary. Thus, substantially identical refers to greater than about 84% identity to another sequence.

  The conjugate of the present invention comprises: a) an antibody or fragment thereof that specifically binds to an ENDO180 polypeptide on the cell surface; and b) an antisense compound, a chemically modified siRNA compound, an unmodified siRN compound, a chemically modified shRNA compound, unmodified A nucleotide-based therapeutic comprising: a nucleotide-based therapeutic selected from a modified shRNA compound, a chemically-modified miRNA compound, and an unmodified miRNA compound; and c) a linking moiety that links (a) to (b) Inhibits the expression of the target gene in the cell.

  A “linker” according to the present invention is a nucleotide or non-nucleotide moiety that links an antibody to a therapeutic molecule. In some embodiments, the linker is a cleavable moiety. Preferred cleavable groups include disulfide bonds, amide bonds, thioamide bonds, ester bonds, thioester bonds, adjacent diol bonds, or hemiacetals. Other cleavable bonds are peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases). Etc., including enzymatically cleavable linkages.

  In some embodiments, the linker is a non-nucleotide linker that includes a peptide linker. The choice of peptide sequence is critical for the success of the conjugate. In some embodiments, the linker is stable to serum proteases but is cleaved by lysosomal enzymes in the target cell. In a non-limiting example, the linker is a peptide selected from the linkers described in US Pat. No. 5,574,142, protamine, fragments of protamine, (Arg) 9, biotin-avidin, biotin-streptavidin, and antennapedia peptides. . For example, peptide linkers are used to link antibodies to nucleotide therapeutics. Other non-nucleotide linkers include alkyl or aryl chains of about 5 to about 100 atoms.

  In some embodiments, the linker is a nucleotide linker. In certain embodiments, the nucleic acid linker has a length in the range of 2-100, preferably 2-50 or 2-30 nucleotides.

Chemical modification “nucleotide” of an oligonucleotide is meant to encompass deoxyribonucleotides and ribonucleotides, which may be natural or synthetic, and / or modified or unmodified. Modifications include changes to the linkage between ribonucleotides in the sugar moiety, base moiety, and / or oligonucleotide. As used herein, the term “ribonucleotide” encompasses natural and synthetic, unmodified and modified ribonucleotides. Modifications include changes to the linkage between ribonucleotides in the sugar moiety, base moiety, and / or oligonucleotide.

  Nucleotides useful for preparing therapeutic agents include naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, adenine, guanine, cytosine, thymine, and uracil. The modified bases of nucleotides are inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyladenines, 5-halouracil, 5-halocytosine, 6-azacytosine and 6-azathymine, pseudouracil 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyladenine, 8-hydroxyl adenine, and other 8-substituted adenines, 8-haloguanine, 8-aminoguanine, 8-thiol Includes guanine, 8-thioalkylguanine, 8-hydroxyguanine, and other substituted guanines, other aza and deazaadenine, other aza and deazaguanine, 5-trifluoromethyluracil, and 5-trifluorocytosine. In some embodiments, one or more nucleotides in the oligomer are replaced with inosine.

According to some embodiments, the present invention provides inhibitory oligonucleotide compounds comprising unmodified and modified nucleotides and / or non-conventional moieties. In certain embodiments, the therapeutic agent is an oligonucleotide. In various preferred embodiments, the therapeutic agent is a double stranded oligonucleotide, preferably an siRNA.

The selection and synthesis of siRNAs corresponding to known genes has been widely reported (eg, Ui-Tei et al., 2006. J Biomed Biotechnol .; 2006: 65052; Chalk et al., 2004. BBRC. 319 (1 ): 264-74; Sioud & Leirdal, 2004.Met.Mol Biol; 252: 457-69; Levenkova et al., 2004, Bioinform.20 (3): 430-2; Ui-Tei et al., 2004.NAR32 ( 3): 936-48).

  For examples of the use and production of modified siRNA, see, eg, Braasch et al. , 2003. Biochem. , 42 (26): 7967-75, Chiu et al. , 2003, RNA, 9 (9): 1034-48, PCT Application Publication Nos. WO 2004/015107 (Atugen AG), and WO 02/44321 (Tuschl et al). U.S. Pat. Nos. 5,898,031 and 6,107,094 teach chemically modified oligomers. US Pat. No. 7,452,987 relates to oligomeric compounds having alternating unmodified and 2 'sugar modified ribonucleotides. US Patent Publication No. 2005/0042647 describes dsRNA compounds having bonds between chemically modified nucleosides.

  Amarzguoui et al. (2003, NAR, 31 (2): 589-595) showed that the activity of siRNA depends on the position of 2'-O-methyl modification. Holen et al (2003, NAR, 31 (9): 2401-2407) showed that siRNA with a small number of 2′-O-methyl modified nucleosides showed good activity compared to the wild type. It has been reported that activity decreased as the number of -O-methyl modified nucleosides decreased. Chiu and Rana (2003, RNA, 9: 1034-1048) each incorporated the 2'-O-methyl modified nucleoside into the sense or antisense strand (fully modified strand) compared to unmodified siRNA. Reported that the siRNA activity was significantly reduced. Although substitutions at the 3 ′ end of the antisense and at both ends of the sense strand were allowed, substitution of the 2′-O-methyl group at the 5 ′ end of the antisense strand was reported to significantly limit activity. (Czauderna et al, 2003, NAR, 31 (11), 2705-2716).

  PCT Patent Application Nos. PCT / IL2008 / 000248 and PCT / IL2008 / 001197, assigned to the assignee of the present invention and incorporated herein by reference in their entirety, are preparations of chemically modified siRNA compounds. Useful motifs are disclosed. PCT Patent Application Publication No. WO2008 / 020435 discloses inhibitors comprising several siRNA compounds against the target genes described herein.

  The compound includes at least one modified nucleotide selected from the group consisting of sugar modification, base modification, and internucleotide linkage modification, and includes DNA, LNA (locked nucleic acid), ENA (ethylene-crosslinked nucleic acid), PNA (peptide nucleic acid). ), Arabinoside, phosphonocarboxylate or phosphinocarboxylate nucleotides (PACE nucleotides), enantiomer nucleotides, or modified nucleotides such as nucleotides with 6 carbon sugars.

  All analogs or modifications of nucleotides / oligonucleotides are used with the present invention, provided that the analogs or modifications do not have a substantially detrimental effect on the function of the nucleotide / oligonucleotide. And Acceptable modifications include sugar moiety modifications, base moiety modifications, internucleotide linkage modifications, and combinations thereof.

Sugar modifications include modifications of the 2 ′ part of the sugar residue and are described in particular as amino, fluoro, alkoxy, eg methoxy, as described in EP 0 586 520 B1 or EP 0 618 925 B1. alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, _thioate, C 1 _{-C 10} lower alkyl, substituted lower alkyl, alkaryl or _{aralkyl, OCF 3, OCN, O-,} S-, or N- alkyl; O-, S-, or N- _{alkenyl; SOCH 3; SO 2 CH 3} ; ONO 2; NO 2, N 3; heterocycloalkyl (heterozycloalkyl); heterocycloalkyl alkaryl (heterozycloalkaryl); aminoalkylamino ; Polyalkyl Amino or including substituted silyl.

  In one embodiment, the siRNA compound comprises at least one ribonucleotide comprising a 2 'modification of the sugar moiety ("2' sugar modification"). In certain embodiments, the compound comprises 2'O-alkyl or 2'-fluoro or 2'O-allyl or any other 2 'modification, optionally at an alternative position. Other stabilizing modifications (eg, terminal modifications) are also possible. In some embodiments, a preferred 2'O-alkyl is a 2'O-methyl (methoxy) sugar modification.

  In some embodiments, the backbone of the oligonucleotide is modified to include phosphate-D-ribose forms, but thiophosphate-D-ribose forms, triesters, thioates, 2′-5 ′ bridged backbones (also 5 ′ -2 '), PACE and the like can also be included.

  As used herein, the term “unpaired nucleotide analog” includes 6 desaminoadenosine (nebulaline), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3- Including, but not limited to, Me Ribo U, N3-Me Ribo T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC Means a nucleotide analog containing a non-base-paired moiety. In some embodiments, the non-base-paired nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide. Polynucleotide analogs may also be prepared, and the structure of one or more nucleotides is radically altered, making them more suitable as therapeutic or experimental reagents. An example of a nucleotide analog is a peptide nucleic acid (PNA) in which the deoxyribose (or ribose) phosphate backbone of DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to enzymatic degradation and have extended stability in vivo and in vitro. Other modifications that can be made to the oligonucleotide include polymer backbone, cyclic backbone, acyclic backbone, thiophosphate-D-ribose backbone, triester backbone, thioate backbone, 2′-5 ′ cross-linked backbone, artificial nucleic acid, morpholino Nucleic acids, glycol nucleic acids (GNA), threose nucleic acids (TNA), arabinosides, and enantiomeric nucleosides (eg, β-L-deoxyribonucleosides instead of β-D-deoxyribonucleosides). Examples of siRNA compounds containing LNA nucleotides are described in Elmen et al. (NAR 2005, 33 (1): 439-447).

  The compounds of the invention can be synthesized using one or more inverted nucleotides, such as inverted thymidine or inverted adenine (see, for example, Takei, et al., 2002, JBC 277 (26): 23800-06). reference).

  Other modifications include terminal modifications of the 5 'and / or 3' portion of the oligonucleotide, also known as the cap portion. Such terminal modifications are selected from nucleotides, modified nucleotides, lipids, peptides, sugars, and inverted abasic moieties.

  In the present invention, what are sometimes referred to as “basic nucleotides” or “basic nucleotide analogs” are more suitably referred to as pseudonucleotides or unconventional moieties. A nucleotide is a monomeric unit of nucleic acid consisting of a ribose or deoxyribose sugar, a phosphate, and a base (adenine, guanine, thymine, or cytosine in DNA, adenine, guanine, uracil, or cytosine in RNA). . Modified nucleotides include modifications at one or more of sugars, phosphates, and / or bases. Abasic pseudonucleotides lack bases and are therefore not strictly nucleotides.

  In some embodiments, the siRNA therapeutic comprises a cap moiety. As used herein, the term “cap moiety” refers to an abasic ribose moiety comprising a 2′O alkyl modification, an abasic deoxyribose moiety, a modified abasic ribose, and an abasic deoxyribose moiety; And abasic deoxyribose moieties and modifications thereof; C6-imino-Pi; enantiomeric nucleotides including L-DNA and L-RNA; 5-Me nucleotides; and 4 ′, 5′-methylene nucleotides; 1- (β- D-erythrofuranosyl) nucleotide; 4′-thionucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; l, 3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; Hexyl phosphate; 12-aminododecyl; hydroxypropyl phosphate; 1,5-anhydrohexitol Α-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3 ′, 4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasic moiety 1,4-butanediol phosphate; 5′-amino; bridged or non-bridged methylphosphonate, and nucleotide analogs containing a 5′-mercapto moiety.

  Certain preferred cap moieties are enantiomer nucleotides including abasic ribose or abasic deoxyribose moieties; inverted abasic ribose or abasic deoxyribose; C6-amino-Pi; L-DNA and L-RNA.

  In some embodiments, the therapeutic siRNA includes moieties other than nucleotides. The term “unconventional moiety” as used herein refers to an abasic ribose moiety, an abasic deoxyribose moiety, a deoxyribonucleotide, a modified deoxyribonucleotide, an enantiomer nucleotide, an abasic paired nucleotide analog, and a 2 ′. Nucleotides linked to adjacent nucleotides by phosphate bonds between 5 ′ nucleotides; refers to cross-linked nucleic acids including LNA and ethylene cross-linked nucleic acids.

  Examples of the abasic deoxyribose moiety include abasic deoxyribose-3′-phosphate, 1,2-dideoxy-D-ribofuranose-3-phosphate, 1,4-anhydro-2-deoxy-D-ribitol- Contains 3-phosphate. Inverted abasic deoxyribose moieties include inverted deoxyribobasic, 3 ', 5' inverted deoxyabasic 5'-phosphate.

  An “enantiomer” nucleotide is a nucleotide that is naturally occurring or has the opposite enantiomer to that of a commonly used nucleotide, ie, a naturally occurring (D-nucleotide) mirror image (L-nucleotide), and In the case of enantiomer ribonucleotides, it is also referred to as L-RNA, and “spiegelmer”. Nucleotides may be ribonucleotides or deoxyribonucleotides and may further comprise at least one sugar, base, and / or backbone modification. See U.S. Patent No. 6,586,238. US Pat. No. 6,602,858 also discloses a nucleic acid catalyst comprising at least one L-nucleotide substitution. Enantiomeric nucleotides include, for example, L-DNA (L-deoxyriboadenosine-3′-phosphate (enantiomer dA); L-deoxyribocytidine-3′-phosphate (enantiomer dC); L-deoxyriboguanosine-3 ′. -Phosphate (enantiomer dG); L-deoxyribothymidine-3'-phosphate (enantiomer dT)) and L-RNA (L-riboadenosine-3'-phosphate (enantiomer rA); L-ribocytidine- 3'-phosphate (enantiomer rC); L-riboguanosine-3'-phosphate (enantiomer rG); L-ribouracil-3'-phosphate (enantiomer dU).

  Modified deoxyribonucleotides are, for example, 5′OMe DNA (5-methyl-deoxyriboguanosine-3′-phosphate); PACE (deoxyriboadenine 3′phosphono), which may be useful as a nucleotide at the 5 ′ terminal position (position number 1). Acetate, deoxyribocytidine 3 ′ phosphonoacetate, deoxyriboguanosine 3 ′ phosphonoacetate, deoxyribothymidine 3 ′ phosphonoacetate.

  The cross-linked nucleic acid is LNA (2′-O, 4′-C-methylene cross-linked nucleic acid adenosine 3 ′ monophosphate, 2′-O, 4′-C-methylene cross-linked nucleic acid 5-methyl-cytidine 3 ′ monophosphate, 2′-O, 4′-C-methylene bridged nucleic acid guanosine 3 ′ monophosphate, 5-methyl-uridine (or thymidine) 3 ′ monophosphate); and ENA (2′-O, 4′-C-ethylene Cross-linked nucleic acid adenosine 3 ′ monophosphate, 2′-O, 4′-C-ethylene cross-linked nucleic acid 5-methyl-cytidine 3 ′ monophosphate, 2′-O, 4′-C-ethylene cross-linked nucleic acid guanosine 3 ′ mono Phosphoric acid, 5-methyl-uridine (or thymidine) 3 ′ monophosphate).

  In some embodiments of the invention, preferred non-conventional moieties are abasic ribose moieties, abasic deoxyribose moieties, deoxyribonucleotides, enantiomer nucleotides, and nucleotides that are linked to adjacent nucleotides by internucleotide phosphate linkages. It is.

  According to one aspect, the present invention provides inhibitory oligonucleotide compounds comprising unmodified and modified nucleotides. The compound includes at least one modified nucleotide selected from the group consisting of sugar modification, base modification, and internucleotide linkage modification, and includes DNA and LNA (locked nucleic acid) including ENA (ethylene-bridged nucleic acid); PNA (peptide Nucleic acid); arabinoside; PACE (phosphonoacetate and its derivatives), enantiomer nucleotides, or modified nucleotides such as nucleotides with 6 carbon sugars. In some embodiments, the present invention provides methods and compositions for inhibiting the expression of target genes in vivo. In general, the method comprises administering a delivery agent-therapeutic agent conjugate. In a specific embodiment, a small interfering RNA (ie, siRNA) that targets mRNA down-regulates the expression of the target gene (reduces mRNA, reduces protein levels) by an RNA interference mechanism. Transcribed from the target gene in sufficient quantity. In particular, the subject method can be used to inhibit the expression of a target gene for the treatment of disease. In accordance with the present invention, siRNA molecules or inhibitors of target genes are used as drugs to treat various pathologies.

  The synthesis of the nucleic acids described in the present invention is within the skill of the artisan. Such a synthesis is described, inter alia, in Beaucage SL and Iyer RP, 1992 Tetrahedron; 48: 2223-2311, Beaucage S. et al. and Iyer RP, 1993 Tetrahedron; 49: 6123-6194, and Caruthers MH et. al, 1987 Methods EnzymoL; 154: 287-313, and the synthesis of thioates is described, inter alia, by Eckstein F. et al. 1985 Annu. Rev. Biochem. 54: 367-402, the synthesis of RNA molecules is described in Sproat B .; , Humana Press 2005 Edited by Herdewijn P .; Kap. 2: 17-31, and each downstream process is described, inter alia, by Pingoud A. et al. et. al. , In IRL Press 1989 Edited by Oliver R. W. A. Kap. 7: 183-208 and Sproat B.R. , In Humana Press 2005 Edited by Herdewijn P. Kap. 2: 17-31 (supra).

  The siRNA for any one of the target genes is synthesized based on the known sequence of the target gene mRNA using methods known in the art described above, and various modifications described herein. To stabilize against serum and / or cellular nucleases.

Target genes The conjugates according to the invention are useful for inhibiting the expression of genes associated with diseases or disorders selected from proliferative diseases, metastatic diseases, and fibrosis.

  Target genes include anti-apoptotic genes, genes related to basic cell division apparatus, genes related to cell cycle control / cell proliferation, genes related to rate-limiting metabolism (nucleotide / nucleic acid synthesis, protein synthesis, energy metabolism), Includes genes associated with protein trafficking (eg secretion), pro-inflammatory genes, cytokines, chemokines, NFkB, growth factors / receptors (TGFβ1 and 2, CTGF, IGF1, PDGF1, PDGF2, VEGF, EGFR, HER2, etc.) .

A non-limiting list of target genes is set forth in Table A below.

Sense and antisense sequences useful in the synthesis of siRNA are selected according to ownership and publicly available methods and algorithms.
The chemical modifications provided above are particularly useful for synthesizing nucleotide therapeutics that exhibit serum stability, activity, reduced immune response, and reduced non-target effects.

The antibody term “antibody” refers to, inter alia, IgG, IgM, IgD, IgA, and IgE antibodies. The definition includes polyclonal or monoclonal antibodies. The term refers to a whole antibody or a fragment of an antibody comprising an antigen binding domain, eg, an antibody that does not include an Fc region, a single chain antibody, a miniantibody, a fragment consisting essentially of the variable antigen binding domain of the antibody. The term “antibody” can also refer to an antibody against a polynucleotide sequence obtained by cDNA vaccination. The term also retains the ability to selectively bind to their antigen or receptor and includes, inter alia, antibody fragments exemplified as follows.
(1) Fab: A fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting the whole antibody with the enzyme papain to produce part of the light and heavy chains.
(2) (Fab ′) 2: A fragment of an antibody that can be obtained without treating the whole antibody with the enzyme pepsin and then reducing it. F (ab′2) is a dimer of two Fab fragments maintained together by two disulfide bonds.
(3) Fv: defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region expressed as two chains.
(4) Single chain antibody (SCA): defined as a genetically engineered molecule containing a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single chain molecule (Including scFv)

  CDR grafting may be performed to modify certain properties of the antibody molecule, including affinity or specificity. Non-limiting examples of CDR grafting are disclosed in US Pat. No. 5,225,539.

  Single domain antibodies are isolated from immunized camelid native heavy chain antibodies including camels and llamas. Small antibodies are very strong and bind antigen with high affinity in monomeric state. US Pat. No. 6,838,254 describes the production of antibodies or fragments thereof derived from camelid heavy chain immunoglobulins.

  Monoclonal antibodies (mAbs) are a substantially homogeneous population of antibodies to a specific antigen. Monoclonal antibodies (mAbs) are obtained by methods known to those skilled in the art. See, for example, Kohler et al (1975); U.S. Patent No. 4,376,110, Ausubel et al (1987-1999), Harlow et al (1988), and Colligan et al (1993). Are incorporated herein by reference in their entirety). The mAbs of the present invention may be any immunoglobulin class mAb, including IgG, IgM, IgE, IgA, and any subclass thereof. The hybridoma producing the mAb may be cultivated in vitro or in vivo. For example, Balb / c mice pre-stimulated with pristine intraperitoneally injected cells from individual hybridomas to produce ascites containing high concentrations of the desired mAb, resulting in high titer mAbs in vivo. It is done. Isotype IgM or IgG mAbs may be purified from such ascites fluid or from culture supernatants using column chromatography methods well known to those skilled in the art.

  “Specific binding affinity” means that an antibody binds to an ENDO180 polypeptide or fragment thereof with greater affinity than it binds to another polypeptide under similar conditions.

  The term “epitope” is intended to refer to a portion of a molecule that can be bound by and also recognized by an antibody. An “antigen” is a molecule or portion of a molecule that can be bound by an antibody and that can further induce an animal to produce an antigen that can bind to an epitope of that antigen. It is. An antigen may have one or more than one epitope. The specific reactions described above are intended to mean that an antigen reacts in a highly selective manner with its corresponding antibody, rather than with many other antibodies that can be triggered by other antigens. The

  Epitopes or antigenic determinants usually consist of chemically active surface molecule groups such as amino acids or sugar side chains and have specific three-dimensional structural characteristics and specific charge characteristics.

  In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the monoclonal antibody is an IgG, IgM, IgD, IgA, or IgE monoclonal antibody. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. In one embodiment, the monoclonal antibody is an IgG monoclonal antibody. In one embodiment, the monoclonal antibody is a human antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the portion of the antibody is a Fab fragment of the antibody. In one embodiment, the portion of the antibody comprises the variable domain of the antibody. In one embodiment, the portion of the antibody comprises the CDR portion of the antibody. In other embodiments, the antibody is an scFv molecule. The antibodies of the present invention may be produced recombinantly (Marshak et al., 1996 "Stratesies for Protein Purification and Characterization. A laboratory course manual." Plainview, N.Y. See generally) analogs may be produced by post-translational processing. Differences in glycosylation can provide polypeptide analogs.

  The antibody can be a human antibody or a non-human antibody. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Methods for humanizing antibodies are well known to those skilled in the art.

  This application provides humanized forms of the above antibodies. As used herein, “humanized” refers to an antibody in which some, most, or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from a human immunoglobulin molecule (eg, , Human framework regions replace non-human regions). In one embodiment of the humanized form of the antibody, some, most, or all amino acids outside the CDR regions are replaced with amino acids from a human immunoglobulin molecule, but some within one or more CDR regions. Most or all amino acids remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible unless the ability to bind antigen ENDO180 is abolished.

  “Humanized” antibodies retain the same antigen specificity as the original antigen, ie, the ability to bind to ENDO180, particularly the human ENDO180 receptor, and are similarly internalized by the receptor.

  Those skilled in the art will know how to produce humanized antibodies of the subject invention. Various publications, some of which are incorporated by reference into this application, describe methods for making humanized antibodies.

  For example, the methods described in US Pat. Nos. 4,816,567 and 6,331,415 involve the production of chimeric antibodies having the variable region of one antibody and the constant region of another antibody.

  US Patent Nos. 5,225,539; 6,548,640 and 6,982,321 differ in immunoglobulin CDRs so that humanized antibodies recognize the target antigen but do not interfere with the immune response. Describes the use of recombinant DNA technology to produce humanized antibodies that are substituted with CDRs from specific immunoglobulins. Specifically, site-directed mutagenesis is used to introduce CDRs onto framework regions.

  Other techniques for humanizing antibodies are described in WO 90/07861 and US Pat. Nos. 5,585,089, 5,693,761, 6,180,370 and 7,022,500. Are described in corresponding patents. These patents describe methods for increasing the affinity of an antibody for a desired antigen by combining the CDRs of a murine monoclonal antibody with the framework and contact regions of a human immunoglobulin. Human framework regions can be selected to maximize homology with mouse sequences. Computer models can be used to identify amino acids within framework regions that may interact with CDRs or specific antigens, and then at these positions, mouse amino acids are used to identify humanized antibodies. Can be created.

  The above methods are merely illustrative of some of the methods that one skilled in the art can utilize to make humanized antibodies.

  Monoclonal antibody E3-8D8 represents an anti-ENDO180 antibody suitable for use in the methods of the invention. The hybridoma cell E3-8D8 was deposited with the Belgian Co-ordinated Collections of Micro-Organisms (BCCM) under the terms of the Budapest Treaty and assigned the accession number LMBP7203CB.

Epitope mapping Epitope mapping studies identify residues that are important for antibody binding. Various methods for epitope mapping are known in the art and are readily implemented by those skilled in the art. A specific method is described in Epitope Mapping: A Practical Approach (OM R. Westwood, FC Hay; Oxford University Press, 2000), which is incorporated herein by reference.

  An example of an epitope mapping technique is a synthetic labeled peptide in which a set of overlapping synthetic peptides, each corresponding to a small section of the linear sequence of protein antigens (ie, the extracellular domain of ENDO180), is synthesized and arranged on a solid phase. Epitope mapping by A panel of peptides is then probed with a test antibody, and a secondary antibody labeled with an enzyme is used to detect the bound antibody.

  Other techniques include fragmentation or cleavage of protein antigens and gel separation, transport to membranes, probing with test antibodies, and detect bound antibodies using a secondary antibody labeled with an enzyme.

Development of antibody pharmaceuticals In general, monoclonal antibodies need to be designed to preserve binding properties (selectivity, internalization, etc.) and reduce the immune response of the recipient. In particular, monoclonal antibodies secreted from hybridoma 3E-8D8 can be optimized as human therapeutics by one of several methods known to those skilled in the art. In one method, the variable heavy chain (V _H ) and variable light chain (V _L ) of a monoclonal antibody are sequenced. Once the amino acid sequence is known, complementarity determining regions (CDRs), heavy and light chain CDR3s are identified, and degenerate oligonucleotides are used to clone synthetic CDR3s into vectors to produce recombinant vectors or constructs To do. The construct can be, for example, a Fab fragment, F (ab ′) 2 fragment, Fv fragment, single chain fragment, or full-length IgG molecule. The construct (s) are expressed and the polypeptide is isolated. In some embodiments, the monoclonal antibody is further optimized by mutagenesis optimized by site-directed mutagenesis to create a CDR3 domain having substantial identity to the original CDR3. May be used.

Therapeutic Agents Therapeutic agents or active agents according to the present invention include nucleotide and non-nucleotide agents, including antisense (AS), miRNA, and oligonucleotides such as unmodified and chemically modified siRNA compounds. Preferred therapeutic agents are siRNA compounds.

  In some embodiments, the siRNA targets the target gene and reduces its expression by RNA interference.

  The therapeutic oligonucleotides of the present invention are synthesized for ribonucleic acid or deoxyribonucleic acid nucleotides by any method known in the art. For example, a commercially available polynucleotide synthesizer (for example, 380B DNA synthesizer manufactured by Applied Biosystems) may be used. Where fragments are used, two or more such sequences may be synthesized and linked for use in the present invention. Although siRNA is a preferred therapeutic agent according to the present invention, the present invention provides that the therapeutic agent is an alkylating agent (such as thiotepa and CYTOXAN® cyclophosphamide); an alkyl sulfonate (such as busulfan, improsulfan and pipersulfan) ); Aridiline (such as benzodopa, carbocone, metredopa, and ureedopa); ethyleneimine and methyl melamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine ); Acetogenin (especially bratacin and butacinone); δ-9-tetrahydrocanabinol (dronabinol, MARINOL®) )); Β lapachone rapacol; colchicine; betulinic acid; camptothecin (including synthetic analogs topotecan (including HYCAMTIN®), CPT-11 (irinotecan, CAMTOSAR®), acetylcamptothecin, scolectin, And 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (including its adzelesin, calzeresin, and bizeresin synthetic analogues); podophyllotoxin; podophyllic acid; teniposide; Cryptophycin 8); cryptophycin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eluterobin; panclastatin; sarcodictin; sponge statin; Nitrogen masters such as busil, chlornafazine, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitine, phenesterine, prednisomine, trophosphamide, uracil mustard; , Lomustine, nimustine, and ranimustine, etc.); antibiotics (eg, Agnew, Chem Intl. Ed. Engl, 1994., etc.); 33: 183-186); dynemicin including dynemicin A; esperamycin; and neocartinostatin luminophore And related chromoprotein ennesin antibiotic luminophores), aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, kactinomycin, carabicin, carminomycin, cardinophyllin, chromomycin, dactinomycin, daunorubicin, detorubicin 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), and Deoxyxorubicin), epirubicin, esorubicin, idarubicin, maceromycin, mitomycin (such as mitomycin C), mycophenolic acid, Ramycin, olivomycin, pepromycin, potomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU), etc .; folic acid analogs (such as denopterin, methotrexate, pteropterin, and trimethrexate); purine analogs (Fludarabine, 6-mercaptopurine, thiampurine, thioguanine, etc.); pyrimidine analogues (ancitabine, azacitidine, 6-azauridine, carmofur) Cytarabine, dideoxyuridine, doxyfluridine, enositabine, floxuridine, etc .; androgen (carsterone, drostanolone propionate, epithiostanol, mepithiostan, test lactone, etc.); anti-adrenal drugs (aminoglutethimide, mitotane, trilostane); folic acid repres Nischer (Floric acid, etc.); Acegraton; Aldophosphamide glycoside; Aminolevulinic acid; Eniluracil; Amsacrine; Vestlabcil; Bisantren; Edatraxate; Defafamine; Demecorsin; Hydroxyurea; lentinan; lonidamine; maytansinoids (maytansine and ansamitocin); mitoguazone; mitoxantrone Nitracrine; pentostatin; phenmet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex; razoxan; rizoxin; schizophyllan; spirogermanium; tenuazonic acid; ', 2 "-t trichloroethyleneamine; trichothecene (especially T-2 toxin, veracrine A, loridine A, and loridine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitoblonitol; Mitractol; pipbloman; gasitocin; arabinoside (“Ara-C”); thiotepa; taxoid (eg, paclitaxel (TAXOL®), pas Albumin engineered nanoparticle formulation of clitaxel (ABRAXONE. TM. ), And doxetaxel (TAXOTERE)); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (cisplatin and carboplatin); vinblastine (VELBAN®); platinum; etoposide (VP-) 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; Difluoromethylolnitine (DMFO); retinoids (such as retinoic acid); pharmaceutically acceptable salts, acids or derivatives of any of the above And combinations of two or more of the above (CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy)) and FOLFOX (oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN ( A conjugate or mixture selected from the abbreviations for treatment plans using

  This definition also includes antihormonal agents that act to control, reduce, block, or inhibit the effects of hormones that can promote cancer growth and are often administered as systemic or systemic therapeutics. included. They may be hormones themselves. Examples include, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxy tamoxifen, trioxyphene, keoxifen, LY11018, onapristone, and toremifene Antiestrogens and selective estrogen receptor modulators (SERM), including (FARESTON®); antiprogesterone; estrogen receptor downregulator (ERD); substances that function to suppress or close the ovary, such as Leuprolide acetate (Luteron-releasing hormone (LHRH) agonists such as LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate, and triptorelin Other antiandrogens such as flutamide, nilutamide, and bicalutamide; and, for example, 4 (5) -imidazole, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (ALOMASIN®), Aromatase inhibitors such as formestani, fadrozole, borozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®), and bisphosphonates such as clodronate (Eg, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), Dronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and troxacitabine (1,3-deoxyribonucleoside cytosine analog) SiRNA, ribozymes and antisense oligonucleotides, particularly those that inhibit gene expression in signal transduction pathways involved in the growth of abnormal cells; vaccines such as THERATOPE® vaccines and gene therapy vaccines, such as ALLOVECTIN® (Trademark) vaccine, LEUVECTIN (R) vaccine, and VAXID (R) vaccine; topoisomerase 1 inhibitors (e.g. LURTOTECAN (R) RmRH (eg, ABARELIX®); lapatinib tosylate (ErbB-2 and EGFR double tyrosine kinase small molecule inhibitor also known as GW572016); celecoxib (CELEBREX®) and other COX- 2-inhibitor; 4- (5- (4-methylphenyl) -3- (trifluoromethyl) -1H-pyrazol-1-yl) benzenesulfonamide; and a pharmaceutical of any of the above Acceptable salts, acids, or derivatives.

  As used herein, the term “polypeptide” refers to peptides and complete proteins in addition to polypeptides, including isolated and recombinant polypeptides. As used herein, “biological function” refers to the biological properties of a molecule and in this context is performed in vivo directly or indirectly by a naturally occurring polypeptide or nucleic acid molecule. By effector or antigen function or activity. Biological functions are: receptor binding, any enzyme activity or enzyme modulating activity, any carrier binding activity, any hormone activity, any internalizing molecule or any activity in translocation from one compartment to another, cell Any activity that promotes or inhibits cell attachment to an outer matrix or cell surface molecule, or any structural role, including having and expressing a nucleic acid sequence encoding a functional protein, It is not limited. Antigen function essentially means possession of an epitope or antigenic site capable of cross-reacting with an antibody raised against a naturally occurring protein. Biologically active analogs share (but need not) the effector function of a natural polypeptide that can retain additional antigenic functions.

  Measurement of the level of ENDO180 polypeptide may be determined by a method selected from the group consisting of immunohistochemistry, Western blotting, ELISA, antibody microarray hybridization, and imaging of target molecules. Such methods are well known in the art, such as, for example, immunohistochemistry, Western blotting, antibody microarray hybridization, and imaging of target molecules.

  Measurement of the level of ENDO180 polynucleotide may be determined, for example, by a method selected from RT-PCR analysis, in situ hybridization, polynucleotide microarray, and Northern blotting. Such methods are well known in the art.

Antisense molecules In some embodiments, the therapeutic agent is an antisense oligonucleotide. The term “antisense” (AS) or “antisense fragment” refers to a polynucleotide fragment (including either deoxyribonucleotides, ribonucleotides, or a mixture of both) having inhibitory antisense activity, as described above. The activity results in a decrease in the expression of the endogenous genomic copy of the corresponding gene. An AS polynucleotide is a polynucleotide comprising contiguous nucleotides having a sequence that is sufficiently long and homologous to a sequence present within the sequence of the gene to allow hybridization of the AS to the target gene. . A number of reviews describe the main aspects of antisense (AS) technology and its therapeutic potential (Aboul-Fadl T., Curr Med Chem. 2005, 12 (19): 2193-214; Crooke ST, Curr MoI Med. 2004, 4 (5): 465-87; Crook ST, Ann Rev Med. 2004, 55: 61-95; Vacek M et al, Cell MoI Life Sci. 2003, 60 (5): 825-33. Cho-Chung YS, Arch Pharm Res. 2003, 26 (3): 183-91, Chemical aspects of AS technology (Crooke et al., Hematol Pathol. 1995, 9 (2): 59-72), cells (Wagner, Nature. 1994, 372 (6504): 333-5) and further reviews on therapeutic aspects (Scanlon, et al, FASEB J. 1995, 9 (13): 1288-96) Specific gene expression. Antisense mediation in can be achieved by the use of modified AS oligonucleotide sequences (for recent reports see Lefbvre-d'Hellencourt et al, 1995; Agrawal, 1996; LevLehman et al, 1997). .

  The AS oligonucleotide sequence may be a short sequence of DNA, usually 15-30 mer, but may be as small as 7-mer (Wagner et al, Nat. Biotech. 1996, 14 (7): 840-4), designed to complement the target mRNA of interest and form an RNA: AS duplex. This duplex formation can prevent the processing, splicing, transport, or translation of the associated mRNA.

  In addition, certain AS nucleotide sequences can induce cellular RNase H activity when hybridized to their target mRNA, causing degradation of the mRNA (Calabretta et al, Semin Oncol. 1996, 23 (1)). : 78-87). In that case, RNase H will cleave the duplex RNA component and potentially release AS to further hybridize with additional molecules of the target RNA. A further mode of action arises from the interaction of AS with genomic DNA, forming a triple helix that can be transcriptionally inactive.

  Sequence target segments of antisense oligonucleotides have the potential that the sequences exhibit favorable energy-related properties that are important for oligonucleotide duplex formation with their complementary templates and have low self-dimerization or self-complementarity Selected to show gender (Anazodo et al, 1996, BBRC. 229: 305-309). For example, the computer program OLIGO (Primer Analysis Software, Version 3.4) is used to determine the melting temperature, free energy properties of antisense sequences, and to estimate potential self-dimerization and self-complementarity properties be able to. The program allows the determination of a qualitative assessment of these two parameters (potential self-dimer formation and self-complementarity), “no possibility” or “some possibility” or “essentially Provides an indicator of “full potential”. Using this program, target segments that have no potential assessment in these parameters are generally selected. However, a segment with “some possibility” in one of the categories may be used. The balance of parameters is used in the selection as is known in the art. In addition, oligonucleotides are also selected as needed so that analog substitutions do not substantially affect function.

  Phosphorothioate antisense oligonucleotides are usually effective in animals and do not show significant toxicity at concentrations that exhibit sufficient pharmacodynamic half-life (Agrawal, et al., PNAS USA. 1997, 94 (6 ): 2620-5), nuclease resistant. Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFGF) with mitogenic and angiogenic properties suppressed glioma cell growth by 80% in a saturating and specific manner (Morrison, J Biol Chem. 1991 266 (2): 728-34). Being hydrophobic, antisense oligonucleotides interact well with phospholipid membranes (Akhter et al., NAR. 1991, 19: 5551-5559). They are in a saturable mechanism that is predicted to be involved in specific receptors following interaction with the cell plasma membrane (Yakubov et al., PNAS, 1989 86 (17): 6454- 58) actively (or passively) transported into living cells (Loke et al., PNAS 1989, 86 (10): 3474-8).

Ribozymes “Ribozymes” are RNA molecules that possess RNA catalytic ability (see Cech for a review) and cleave specific sites in target RNAs. In accordance with the present invention, ribozymes that cleave mRNA may be used as therapeutic agents. This may be necessary when antisense therapy is limited due to stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes that target genes associated with bone marrow disease may then be used. The number of RNA molecules cleaved by the ribozyme is greater than expected by stoichiometry (Hampel and Tritz, Biochem. 1989, 28 (12): 4929-33; Uhlenbeck, Nature. 1987.328 (6131): 596-600). Ribozymes catalyze the phosphodiester bond cleavage of RNA. Several ribozyme structural families have been identified, including group I nitrones, RNase P, hepatitis delta virus ribozymes, hammerhead ribozymes, and hairpin ribozymes originally derived from the negative strand of tobacco ring spot virus satellite RNA (sTRSV). (US Pat. No. 5,225,347). The latter two families are derived from viroids and virusoids, where ribozymes are thought to separate monomers from oligomers made during rolling circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleaving mRNAs for gene therapy (Sullivan, 1994). In general, ribozymes have a length of about 30-100 nucleotides. Ribozyme delivery is similar to AS fragment and / or siRNA molecule delivery.

siRNA and RNA Interference RNA interference (RNAi) is a phenomenon involved in gene-specific post-transcriptional silencing that relies on duplex (ds) RNA. Early attempts to study this phenomenon and experimentally manipulate mammalian cells were active, non-specific antiviral defense mechanisms activated in response to long dsRNA molecules (Gil et al., Apoptosis, 2000.5: 107-114). It was later discovered that synthetic duplexes of 21 nucleotide RNAs can mediate gene-specific RNAi in mammalian cells without stimulating general antiviral defense mechanisms (Elbashir et al. Nature). 2001, 411: 494-498 and Caplen et al., PNAS 2001, 98: 9742-9747). As a result, short interfering RNA (siRNA), a short double-stranded RNA, has been widely used to inhibit gene expression and understand gene function.

  RNA interference (RNAi) is a small interfering RNA (siRNA) (Fire et al, Nature 1998, 391: 806) or microRNA (miRNA) (Ambros V. Nature 2004, 431: 350-355), and Bartel DP. Cell. 2004 116 (2): 281-97). The corresponding process is commonly referred to as specific post-transcriptional gene silencing when observed in plants and querying when observed in fungi.

  An siRNA is a double-stranded RNA that down-regulates or silences (ie, completely or partially inhibits) the expression of an endogenous or exogenous gene / mRNA. RNA interference is based on the ability of specific dsRNA species to enter specific protein complexes, which then target and specifically degrade or cleave complementary cellular RNA (ie, mRNA). . Thus, an RNA interference response involves siRNA, commonly referred to as RNA-induced silencing complex (RISC), that mediates the cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Features an endonuclease complex containing. Cleavage of the target RNA can occur in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir, et al., Genes Dev., 2001, 15: 188). More specifically, longer dsRNA is digested by type III RNAse into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNA or “siRNA”) (DICER, DROSHA, etc. (Bernstein) et al., Nature, 2001, 409: 363-6 and Lee et al., Nature, 2003, 425: 415-9) The RISC protein complex recognizes these fragments and complementary mRNAs. The entire process is terminated by endonuclease cleavage of the target mRNA (McManus and Sharp, Nature Rev Genet, 2002, 3: 737-47, Paddison and Hannon, Curr Opin Mol Ther. 2003, 5 (3): 217-24) (For further information regarding these terms and proposed mechanisms, see, eg, Bernstein, et al., RNA. 2001, 7 (11): 1509-21, Nishikura, Cell. 2001, 107 (4): 415-8 and PCT Publication No. WO 01/36646).

  Studies have shown that siRNA can be effective in vivo in mammals including humans. Specifically, Bitko et al. Have been shown to be effective in the treatment of mice when a specific siRNA targeting the respiratory syncytial virus (RSV) nucleocapsid N gene is administered intranasally (Nat. Med. 2005, 11). (1): 50-55). For reviews of therapeutic applications of siRNA, see, for example, Barik (Mol. Med 2005, 83: 764-773) and Chakraborty (Current Drug Targets 2007 8 (3): 469-82). In addition, clinical studies using short siRNA targeting the VEGFR1 receptor have been performed in human patients to treat age-related macular degeneration (AMD) (Kaiser, Am J Ophthalmol. 2006 142 (4): 660-8). For more information on the use of siRNA as therapeutic agents, see Durcan, 2008. Mol. Pharma. 5 (4): 559-566, Kim and Rossi, 2008. BioTechniques 44: 613-616, Grimm and Kay, 2007, JCI, 117 (12): 3633-41.

  The siRNA according to the present invention is unmodified, recombinant or chemically modified. Examples of chemical modifications useful in the synthesis of siRNA are disclosed in PCT Patent Application Publication No. WO 2009/044392, which is assigned to the assignee of the present invention and incorporated herein by reference in its entirety.

Pharmaceutical Composition The present invention provides a pharmaceutical composition comprising any one of the above compounds and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition according to the present invention comprises:
a) a monoclonal antibody produced by the hybridoma cell line E3-8D8 (BCCM accession number LMBP 7203CB) b) an antibody or fragment thereof that binds to the same epitope as the antibody of (a),
c) the humanized antibody of (a) or (b),
d) a recombinant polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6 or a variant thereof, and e) a recombinant polypeptide comprising CDR3 having the amino acid sequence set forth in SEQ ID NOs: 7 and 8, or a variant thereof,
f) an anti-ENDO180 antibody or antigen-binding fragment thereof selected from the conjugates of any one of (a) to (e) above conjugated to a moiety,
An antibody or antigen-binding fragment thereof, wherein upon contact with a cell expressing ENDO180, the antibody or antigen-binding fragment thereof is internalized into the cell;
A pharmaceutically acceptable vehicle or carrier.

  In some embodiments, the carrier comprises lipid particles or lipidated glycosaminoglycans.

  Another aspect of the invention provides a compound comprising a) an anti-ENDO180 antibody or antigen-binding fragment thereof, b) a moiety selected from a detectable label, cytotoxic agent, or therapeutic agent, and c) a nanocarrier. To do.

In various embodiments, the nanocarrier is a polysaccharide-based nanoparticle. In various embodiments, the trimolecular compound of the present invention comprises
Formula A-X-Y
X-A-Y or XY A can be represented by one of
Where A represents a detectable label, cytotoxic agent, or therapeutic agent;
X represents a nanocarrier,
Y represents an anti-ENDO180 antibody or an antigen-binding fragment thereof.

  The disclosed compounds are specific cells or tissues that express the ENDO180 polypeptide such that the detectable label, cytotoxic agent, or therapeutic agent is delivered to the desired cell with greater effectiveness and specificity. Designed to target. For example, one embodiment of the present disclosure includes compounds that target cancerous cells and / or tissues.

  As such, specific examples of these compounds include anti-ENDO180 antibodies or antigen-binding fragments thereof that bind to the ENDO180 receptor present on cancer cells at higher concentrations than normal cells. Embodiments of the disclosed compounds utilize upregulation of ENDO180 receptor expression in diseased cells and tissues to selectively deliver therapeutic agents to such cells.

  In various embodiments, the nanocarrier is a polysaccharide-based nanoparticle. In certain embodiments, the polysaccharide is a glycosaminoglycan or a mucopolysaccharide. In various embodiments, the glycosaminoglycan is selected from the group consisting of, but not limited to hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and combinations thereof.

  In certain embodiments, the nanocarrier comprises an organic polymer that includes, but is not limited to, an organic polymer that self-assembles to form self-assembled nanoparticles that provide species that are efficiently multivalent. In such embodiments, the self-assembled nanoparticles can include the same or different compounds. For example, self-assembled nanoparticles include a compound having an anti-ENDO180 antibody or antigen-binding fragment thereof, a moiety selected from a detectable label, cytotoxic agent, or therapeutic agent, and a nanocarrier component.

  Embodiments of the disclosed compounds include a plurality of therapeutic agents, detectable labels for cytotoxic agents. In such embodiments, the compound comprises different therapeutic agents, contrast agents, and cytotoxic agents. In certain embodiments, compounds having more than one therapeutic agent have an increased therapeutic effect due to multivalent effects.

  The present invention further provides pharmaceutical compositions comprising the disclosed compounds and conjugates formulated for administration to a subject. A further aspect of the invention provides a method of treating a subject having a proliferative disease, including cancer, metastatic disease, and fibrosis, using the disclosed compounds, and thus pharmaceutical compositions are provided for this purpose. Is provided herein for

  In a preferred embodiment, the therapeutic agent is a chemically modified siRNA compound that inhibits the expression of the target gene listed in Table A.

  In some embodiments, the composition comprises a mixture of two or more different therapeutic agents, including two or more siRNAs that target a single gene or multiple genes.

  The present invention further comprises at least one compound of the present invention that binds covalently or non-covalently to one or more compounds of the present invention in an amount effective to inhibit the expression or activity of the target gene; A pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier.

  The pharmaceutically “effective amount” for purposes herein is thus determined by such considerations as are known in the art. Amounts to achieve improvements including, but not limited to, improvement in survival or faster recovery, or improvement or elimination of symptoms, and other indicators selected as appropriate measures by those skilled in the art Must be valid. The compounds of the present invention can be administered by any of the conventional routes of administration. The compounds may be administered as compounds or pharmaceutically acceptable salts, and as active ingredients alone or in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants, and vehicles. Note that it may be administered. The compounds can be administered orally, subcutaneously or parenterally and include intravenous, intraarterial, intramuscular, intraperitoneal and intranasal administration, as well as intrathecal and injection procedures. Also useful are grafts of compounds. Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parenteral routes of administration. Liquid compositions include aqueous solutions with or without organic cosolvents, aqueous or oily suspensions, emulsions with edible oils, and similar pharmaceutical vehicles. Also, under certain circumstances, the composition for use in the novel treatment of the present invention may be formed as an aerosol for intranasal administration or the like. The patient to be treated is a warm-blooded animal, in particular a mammal including mankind. Pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants, and vehicles, as well as transplant carriers are inert, non-toxic solid or liquid filters, diluents that do not react with the active ingredients of the invention, Or generally refers to encapsulated materials, which include liposomes, lipidated glycosaminoglycans, and microspheres. Examples of delivery systems useful in the present invention include US Pat. Nos. 5,225,182, 5,169,383, 5,167,616, 4,959,217, 4, 925,678, 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475 196. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

  In general, the active dose of a compound for human use will be a regimen of 1 time per day, or 2 or 3 times per day or more over a period of 1-2 weeks or longer, preferably 24 to 48 hours. Or by continuous infusion for a period of 1 to 2 weeks or longer, preferably in the range of about 1 mg to about 20-100 mg / kg body weight per day, preferably about 0.01 mg / kg to about 2-10 mg / kg body weight per day It is.

  Furthermore, the present invention provides a method of inhibiting the expression of a gene of the present invention by at least 50% compared to a control comprising contacting an mRNA transcript of the gene of the present invention with one or more of the compounds of the present invention. To do.

  In one embodiment, the therapeutic agent inhibits the target gene, and the inhibition is selected from the group consisting of inhibition of gene function, inhibition of polypeptide, and inhibition of mRNA expression.

  The pharmaceutical composition is formulated to provide a single dose or multiple doses.

  In various embodiments, a pharmaceutical composition comprising a conjugate or mixture of the invention is administered to a subject intravenously, intramuscularly, topically, or subcutaneously.

  The pharmaceutical compositions according to the present invention can also be used in a method for preventing and / or treating a disease disclosed herein, which method comprises any of the diseases described herein. For this purpose, the administration comprises a conjugate according to the invention, a mixture according to the invention, or a pharmaceutical composition or drug according to the invention.

Diagnosis The compounds of the present invention are useful for diagnosing cells expressing ENDO180 in a biological sample.

Delivery In some embodiments, antibodies, antigen-binding fragments, and / or conjugates of the invention are targeted by direct application of a naked molecule prepared using a carrier or diluent. Delivered to the tissue.

  The term “naked molecule” includes any delivery vehicle that acts to assist, facilitate, or facilitate entry into the cell, including viral sequences, viral particles, lipid particles, liposomal formulations, lipofectin, or precipitants, etc. No antibody, antigen-binding fragment, or conjugate. For example, siRNA in PBS is “naked siRNA”. However, in some embodiments, the antibodies, antigen-binding fragments, or conjugates of the invention are delivered with lipid particles, polysaccharide particles, or combinations thereof, liposomal formulations, lipofectin formulations, etc., and are well known to those skilled in the art. It can be prepared by a method. Such methods are described, for example, in US Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

  In other embodiments, an antibody, antigen-binding fragment, or conjugate of the invention is attached to or encapsulated within a delivery particle. In some embodiments, the ENDO180 binding domain of the conjugate molecule is exposed to the outer surface of the delivery particle. In some embodiments, the delivery particle is a liposome. In certain preferred embodiments, the delivery particles are lipidated glycosaminoglycan particles. Such particles are described, for example, in US Patent Application No. 10 / 487,022 (Application No. 200402241248), US Patent Application No. 11 / 718,485 (Application Publication Number), which is incorporated herein by reference. No. 20080248092), US patent application Ser. No. 11 / 632,647 (Application No. 20090022656). Without wishing to be bound by theory, the antibody or antigen-binding fragment thereof is exposed on the surface of the delivery particle and travels toward the target cell expressing the ENDO180 polypeptide on that surface, ie, the target and To do.

  Pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants, and vehicles, as well as transplant carriers are inert, non-toxic solid or liquid filters, diluents that do not react with the active ingredients of the invention, Or, generally refers to encapsulated materials, which include liposomes and microspheres. Examples of delivery systems useful in the present invention include US Pat. Nos. 5,225,182, 5,169,383, 5,167,616, 4,959,217, 4, 925,678, 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475 196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. In one particular embodiment of the present invention, topical and transdermal formulations may be selected. The siRNA or pharmaceutical composition of the present invention can be used to determine the clinical status of an individual patient, the disease being treated, the site and method of administration, the schedule of administration, the patient's age, gender, weight, and other known to the healthcare professional. Taking into account factors, it is administered and dosed according to medical practice standards.

  A “therapeutically effective amount” for purposes herein is thus determined by such considerations as are known in the art. Dosages to achieve improvements including, but not limited to, improved survival or faster recovery, or improved or eliminated symptoms, and other indicators selected as appropriate measures by those skilled in the art Must be valid.

  In general, the active dose of a compound for humans is 1 kg per day per kg of body weight in a regimen of 1 time per day, or 2 or 3 times per day or more over a period of 1 to 4 weeks or longer. 1 ng to about 20-100 mg, preferably in the range of about 0.01 mg to about 2-10 mg per kg body weight per day.

  The compounds of the present invention can be administered by any of the conventional routes of administration. The compound may be administered as a compound or pharmaceutically acceptable salt, alone or in combination with a pharmaceutically acceptable carrier, solvent, diluent, excipient, adjuvant, and vehicle. Note that may be administered as well. The compounds can be administered orally, subcutaneously, or parenterally, including intravenous, intraarterial, intramuscular, intraperitoneal, and intranasal, aspiration, transtympanic, and intrathecal and injection procedures. Including. Also useful are grafts of compounds. Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, transtympanic, nasal, and other parenteral routes of administration. Liquid compositions include aqueous solutions with or without organic cosolvents, aqueous or oily suspensions, emulsions with edible oils, and similar pharmaceutical vehicles. In certain embodiments, administration includes intravenous administration. In another embodiment, administration includes local or local administration. In certain embodiments, the composition for use in the novel treatments of the invention may also be formed as an aerosol, eg, for intranasal administration.

In certain embodiments, oral compositions (tablets, suspensions, solutions, etc.) such as oral compositions suitable for mouth washes for the treatment of oral mucositis can be effective for local delivery to the oral cavity.

  Liquid forms are prepared for drops or propellants. Liquid compositions include aqueous solutions with or without organic cosolvents, aqueous or oily suspensions, emulsions with oils, and similar pharmaceutical vehicles. In some embodiments, administration includes local or local administration.

  These compounds can be administered by any suitable route to the eye using, for example, sprays or drops, and topically using ointments, suspensions, or drops, humans and others for treatment. Of animals.

  In a preferred embodiment, the subject to be treated is a warm-blooded animal, specifically a mammal including a human.

  Suitable methods for delivery of the compounds of the invention include systemic delivery, transfection, lipofection, and electroporation, among others. In a further aspect, the invention provides a delivery molecule-therapeutic agent conjugate or anti-ENDO180 antibody or anti-ENDO180 antibody-therapeutic mixture according to the invention and a pharmaceutically acceptable carrier, diluent or adjuvant, or other vehicle. Relates to a pharmaceutical composition comprising (s).

  Preferably, such carriers, diluents, adjuvants, and vehicles are inert and nontoxic. The pharmaceutical composition is adapted for various modes of administration in its various embodiments. Such administration includes systemic and topical administration, as well as oral, subcutaneous, parenteral, intravenous, intraarterial, intramuscular, intraperitoneal, intranasal, intrathecal, transtympanic, and intraocular.

  In some embodiments, the vehicle is selected from lipid particles, polysaccharide particles, or combinations thereof, and lipidated glycosaminoglycan particles (gagomers). In various embodiments, the delivery molecule-therapeutic agent conjugate or antibody-therapeutic agent mixture is at least partially exposed to the outer surface of the lipid particle or lipidated glycosaminoglycan particle.

  Those skilled in the art will recognize that the pharmaceutical composition and the amount of each siRNA may vary depending on the clinical condition of the individual patient, the site and method of administration, the schedule of administration, the patient's age, gender, weight, and other known to the healthcare professional. You will recognize that it depends on the factors. A pharmaceutically effective amount for prophylactic and / or therapeutic purposes is thus determined by such considerations as are known in the art. Preferably, the amount includes improving the condition or providing a faster recovery, improvement or elimination of symptoms and other indicators selected as appropriate measures by those skilled in the medical arts, It is effective to achieve an improvement not limited to this.

Combination therapy In various embodiments, the present invention relates to combination therapy. In one embodiment, co-administration of two or more therapeutic agents achieves a synergistic effect, ie, a therapeutic effect that is higher than the sum of the therapeutic effects of the individual components of the combination. In another embodiment, simultaneous administration of two or more therapeutic agents achieves an additional effect.

  The active ingredients that make up the combination therapy may be administered together by a single dosage form or may be administered by separate administration of each active agent. In certain embodiments, the first and second therapeutic agents are administered in a single dosage form. The drug may be formulated into a single tablet, pill, capsule, or solution, such as for parenteral administration. Alternatively, the first therapeutic agent and the second therapeutic agent may be administered as separate compositions. The first active agent may be administered at the same time as the second active agent, or the first active agent may be administered intermittently with the second active agent. The length of time between administration of the first therapeutic agent and the second therapeutic agent can be adjusted to achieve the desired therapeutic effect. For example, the second therapeutic agent can be only a few minutes (eg, 1, 2, 5, 10, 30, or 60 minutes) or hours (eg 2, 4, 6, 6) after administration of the first therapeutic agent. 10, 12, 24, or 36 hours). In certain embodiments, it may be advantageous to administer one or more of the therapeutic agents more than once during the administration of the second therapeutic agent. For example, the second therapeutic agent may be administered 2 hours after administration of the first therapeutic agent and then again 10 hours. Alternatively, in certain embodiments, it may be advantageous to administer the first therapeutic agent one or more times during administration of the second therapeutic agent. Importantly, the therapeutic effect of each active ingredient overlaps for at least part of the duration of each therapeutic agent, such that the overall therapeutic effect of the combination therapy is due in part to the combined or synergistic effects of the combination therapy That is desirable.

  The present invention relates to compounds and use of compounds that down regulate the expression of the genes of the present invention, and in particular to conjugates comprising low interfering RNA (siRNA). Methods, molecules, and compositions useful for target gene inhibition are detailed herein and any of the molecules and / or compositions described above can be used to treat a subject suffering from a proliferative or fibrotic disease. Can be beneficially used.

Methods of treatment Further aspects of the invention provide methods of treating proliferative diseases, including cancer, metastatic disease, and fibrosis. Methods for the treatment of diseases or disorders associated with uncontrollable pathological cell growth (eg cancer, psoriasis, autoimmune diseases), especially diseases associated with ischemia and lack of adequate blood flow (eg myocardium Methods for the treatment of infarction (MI) and stroke) are provided. In particular, the compounds and compositions of the present invention are useful in the treatment of proliferative diseases in which ENDO180 is expressed in at least some of the diseased cells and / or tissues.

  “Cancer” or “tumor” refers to an abnormal growth of cells. These terms include both primary tumors, which can be benign or malignant, and secondary tumors or metastases that have spread to other parts of the body. Examples of proliferative diseases include, among others, cell tumors (eg, breast, colon, and lung), leukemias such as B cell leukemia, lymphomas such as B cell lymphoma, blastomas such as neuroblastoma, and melanoma and Includes sarcoma. That the pharmaceutical composition according to the invention can be used for any of the diseases associated with undesired blood vessel development or growth, including angiogenesis, and any of the diseases and conditions described herein. I want to be recognized.

  The present invention relates to a cancerous proliferative disease in which cancer cells express the ENDO180 polypeptide (eg, lung cancer, breast cancer, cervical cancer, colon cancer, stomach cancer, kidney cancer, leukemia, liver cancer, lymphoma, ovarian cancer, pancreatic cancer, prostate Methods and compositions are provided for treating patients suffering from cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, and uterine cancer. In one particular embodiment, the cancer is renal cancer, including RCC and TCC.

  “Cancer” and “cancerous disease” are used interchangeably and refer to diseases caused by or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. . Examples of cancerous diseases include leukemia (eg, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia , Acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and sarcoma and Solid tumors such as carcinomas (eg fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, hemangiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphatic endothelial sarcoma, synovial tumor, mesothelioma , Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary gland Cancer, cystadenocarcinoma Medullary cancer, primary bronchial cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonic cancer, Wilms tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer , Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland, hemangioblastoma, acoustic nerve tumor, oligodenroglioma, Schwannoma, meningioma , Melanoma, neuroblastoma and retinoblastoma). Includes metastasis of primary cancer. In some preferred embodiments, the compounds of the invention are useful for treating renal cancer, breast cancer, ovarian cancer, and their metastasis in various organs including lung and bone.

  As used herein, the term “proliferative disorder” refers to any disorder in which cell growth, either malignant or benign, contributes to the condition. Such unwanted growth is characteristic of cancer and many chronic inflammatory diseases, so examples of “proliferative diseases” include the cancers listed above, as well as psoriasis, inflammatory bowel disease, rheumatoid arthritis, etc. Chronic inflammatory proliferative diseases, proliferative cardiovascular diseases such as restenosis; proliferative eye disorders such as diabetic retinopathy; and benign hyperproliferative diseases such as hemangiomas.

Fibrotic diseases Fibrotic diseases are a group of chronic diseases characterized by excessive production of fibrous substances called extracellular matrix that contribute to abnormal changes in tissue structure and interfere with normal organ function. It is. Millions of people around the world suffer from these chronic diseases that are often life threatening. Unfortunately, although fibrosis is widespread, debilitating and often life threatening, there are currently no effective treatments available.

  The human body responds to trauma and injury from scarring. Fibrosis, a disease characterized by excessive scarring, occurs when the normal wound healing response is disturbed. During fibrosis, the wound healing response continues to cause excessive production and precipitation of collagen.

  Although fibrotic disorders can be acute or chronic, the disorders share a common feature of excessive collagen accumulation and associated loss of function when normal tissue is replaced with scar tissue.

  Fibrosis results from a variety of causes and can be identified in various organs. Cirrhosis, pulmonary fibrosis, sarcoidosis, keloids, hypertension, and renal fibrosis are all chronic diseases that induce progressive fibrosis that causes a continuous loss of tissue function.

  Acute fibrosis (usually with a sudden and severe onset and short duration) can result in various forms of trauma, infection, surgery (heart), including accidental damage (especially damage to the spine and central nervous system). It occurs as a common response to post-attack heart scarring), burns, environmental pollution, alcohol and other types of toxins, acute respiratory distress syndrome, radiation, and chemotherapy treatment. All tissues damaged by trauma tend to scar and become fibrous, especially when the damage is repeated. Severe organ fibrosis is often very serious because progressive loss of organ function leads to morbidity, hospitalization, dialysis, disability, and even death. Fibrous diseases or disorders where fibrosis is evident are pulmonary fibrosis, interstitial lung disease, human fibrotic lung disease, liver fibrosis, cardiac fibrosis, macular degeneration, retina and vitreous retinopathy Including inflammatory bowel disease, including myocardial fibrosis, Graves' ophthalmopathy, drug-induced ergot poisoning, cardiovascular disease, atherosclerosis / restenosis, keloid and hypertrophic scar, leprosy, and collagenous colitis.

  Further information on different types of fibrosis can be found in, for example, Yu et al (2002), “Therapeutic strategies to halt real fibrosis”, Curr Opin Pharmacol. 2 (2): 177-81, Keane and Lyle (2003), “Recent advance in management of type 2 diabetics and nephropathy: lessons from the RENAAL study”, Am J Didne. 41 (3 Suppl 2): S22-5, Bohle et al (1989), “The pathogenesis of chronic ral failure”, Pathol Res Pract. 185 (4): 421-40, Kikkawa et al (1997), "Mechanism of the progression of diabetic nephropathy to real failure", Kidney Int Suppl. 62: S39-40, Batler and Brenner (2001), “Hepatic stellate cells as a target for the treatment of liver fibrosis”, Semin Liver Dis. 21 (3): 437-51, Gross and Hunningake (2001) "Idiopathic primary fibrosis", N Engl J Med. 345 (7): 517-25, Frohlich (2001) "Fibrosis and ischemia: the real risks in hypersensitive disease", Am J Hypertens; 14 (6 Pt 2): 194S-199S.

Diabetic nephropathy Diabetic nephropathy, characterized by glomerulosclerosis and renal fibrosis, is the single largest epidemic of end-stage renal disease in modern society, and diabetics have the largest dialysis population. Configure. Such treatment is costly and far from optimal. Transplantation provides a good prognosis, but donor shortages are serious. Because most of the molecular mechanisms underlying these pathologies are unknown, no specific treatment has been developed for diabetic nephropathy (and other types of kidney conditions). Identification of essential functional target genes that are modulated in disease and affect the prognostic severity of diabetic nephropathy has high diagnostic and therapeutic value.

It is known in the art that many pathological processes in the kidney eventually lead to similar or identical morphological changes, namely glomerulosclerosis and fibrosis. Human kidney disease can originate from a variety of sources, including glomerulonephritis, nephritis associated with systemic lupus, cancer, physical obstruction, toxins, metabolic diseases, and immunological diseases, which are All result in renal fibrosis. The implication of this phenomenon is that the same single type of injury results in fibroblast proliferation, two characteristics of fibrosis, and overproduction of various protein components of connective tissue by fibroblasts. It is to be concentrated in the genetic program. In addition, thickening of the basement membrane in the glomerulus accompanies interstitial fibrosis, leading to glomerulosclerosis. Genes encoding proteins involved in renal fibrosis and glomerulosclerosis can be roughly divided into two groups.
1. A gene whose expression triggers the proliferation of fibroblasts, and its expression leads to overproduction of various protein components of connective tissue. These can be specific for different pathologies.
2. A gene whose expression results in the execution of a “fibrous or sclerosing program”. These can be common with all renal pathologies leading to fibrosis and glomerulosclerosis.

  Identification of genes belonging to the second group should contribute to the understanding of the molecular mechanisms involving proliferation and hypersecretion of fibroblasts and mesangial cells, for drug discovery aimed at preventing renal failure. A target gene can be constructed. Application of such agents is expected to suppress, delay, prevent, inhibit, or reduce the progression of fibrosis and glomerulosclerosis.

Combination Therapy The present invention provides combination therapy for the proliferative diseases described in the present invention, particularly cancer. In the combination therapy, one or more genes are targeted to ameliorate the symptoms of the disease being treated. These genes are inhibited by the antibody-nucleotide complex of the present invention.

Kits Further provided are kits comprising at least one anti-ENDO180 monoclonal antibody of the present invention. “Kit” refers to any product (eg, package or container) that contains at least one reagent for specific binding to ENDO180, ie, an antibody. The kit can be used to perform the methods of the invention including therapeutic treatment and diagnosis. In addition, the kit may include a package insert describing the kit, its contents, and usage.

In one embodiment, the kit of the present invention comprises
a) Monoclonal antibody produced by the hybridoma cell line E3-8D8 (BCCM accession number LMBP 7203CB) b) An antibody or fragment thereof that binds to the same epitope as the antibody of (a) c) Substantially similar to SEQ ID NO: 6 A fragment of an antibody comprising a polypeptide d) a recombinant polypeptide comprising a CDR having an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NOs: 7 and 8;
At least a composition comprising an anti-ENDO180 antibody or antigen-binding fragment thereof selected from

  While the present invention has been described in an exemplary manner, it is to be understood that the terminology used has been intended to be descriptive in nature rather than limiting.

Citation of any reference in this specification shall be such that such reference is appropriate prior art or is considered for the patentability of any claim of the present invention. It is not intended for approval. Any statement regarding the content or date of any document is based on information available to the applicant at the time of filing and does not constitute an admission as to the validity of such description.

General Methods in Molecular Biology For standard molecular biology techniques known in the art and not specified herein, see generally Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989), and Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), and Perbal, A Practical Guide to Molecular Cloning, John & Won. , Recombinant DNA, Scientific American Books, New York, and In Birren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998), and U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, And in accordance with the methodology described in US Pat. No. 5,272,057, which are incorporated herein by reference. Polymerase chain reaction (PCR) was performed as generally described in the PCR protocol A Guide To Methods And Applications, Academic Press, San Diego, CA (1990). In situ (intracellular) PCR combined with flow cytometry can be used to detect cells containing specific DNA and mRNA sequences (Testoni et al, 1996, Blood 87: 3822).

  General Methods in Immunology: Standard methods in immunology that are known in the art and not specified herein are generally those of Stites et al (eds), Basic and Clinical Immunology (8th Edition), Appleton & Language. , Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W .; H. Freeman and Co. , New York (1980).

  Immunoassays: ELISA immunoassays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in this assay. If desired, other immunoassays such as radioimmunoassay (RIA) may be used as known to those skilled in the art. Available immunoassays are extensively described in the patent and scientific literature. For example, U.S. Pat. Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517 No. 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771, and 5,281,521, and Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, New York, 1989.

Antibody Production As used herein, the term “antibody” refers to both polyclonal and monoclonal whole antibodies capable of binding to epitope determinants, and fragments thereof (Fab, F (ab ′) 2, scFv). , And Fv). These antibody fragments retain the ability to selectively bind with its antigen or receptor, and are exemplified as follows.
(1) Fab: A fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be produced by digesting the whole antibody with the enzyme papain to produce part of the light and heavy chains.
(2) (Fab ′) 2: A fragment of an antibody that can be obtained without treating the whole antibody with the enzyme pepsin and then reducing it. F (ab′2) is a dimer of two Fab fragments maintained together by two disulfide bonds.
(3) Fv: defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region expressed as two chains.
(4) scFv (single chain antibody (SCA)): genetically engineered containing a light chain variable region and a heavy chain variable region linked by a suitable polypeptide linker as a genetically fused single chain molecule Defined as a molecule.

  Such fragments having the functional activity of an antibody can be prepared by methods known to those skilled in the art (Bird et al. (1988) Science 242: 423-426).

  Conveniently, antibodies can be prepared against the immunogen or part thereof, eg synthetic peptides prepared by sequence-based or recombinantly by clonal techniques, or natural gene products and / or Some of them may be isolated and used as immunogens. Harlow and Lane (1988), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Borrebaeck (1992), Antibodic EnG. H. Freeman and Co. , NY, as generally described, can be used to produce antibodies by standard antibody production techniques well known to those skilled in the art.

  To produce polyclonal antibodies, a host such as a rabbit or goat is immunized with an immunogen or immunogen fragment, usually with an adjuvant and optionally attached to a carrier, and the antibody against the immunogen is recovered from the serum. To do. Furthermore, polyclonal antibodies can be absorbed to monospecificity, i.e., by absorbing serum against the relevant immunogen so that no cross-reacting antibody remains in the serum, the antibody Is monospecific.

  To generate monoclonal antibodies, the technique involves hyperimmunization of an appropriate donor (generally a mouse) with an immunogen and isolation of spleen antibody-producing cells. These cells are fused with immortal cells such as myeloma cells to provide an immortal, fused cell hybrid that secretes the necessary antibodies. The cells are then cultured in large quantities and the monoclonal antibody is harvested from the culture medium and used.

  In order to produce recombinant antibodies, Huston et al. (1991) "Protein engineering of single-chain Fv analogs and fusion proteins" in Methods in Enrology (JJ Langone, ed., Nand Y, 1919, Academic Press, New York, 1994). single-chain Fvb derivatives of monoclonal antigens and ther production in Escherichia coli in Methods in Enzymology, JJ Langeon, NJ. , NY) 203: 88-99, Mernaugh and Mernaugh (1995) "An overview of Phage-displayed Recombinant Antibodies" in Molecular Methods In Plant RP. 359-365) and messenger RNA from animal antibody-producing B lymphocytes or hybridomas can be reverse transcribed to obtain complementary DNA (cDNA). Can be full length or partial length, amplified and cloned in a phage or plasmid. The antibody or antibody fragment can be expressed using a suitable expression system to obtain a recombinant antibody, which can also be separated or connected by It can also be obtained by screening an appropriate expression library.

  As is well known in the art, an antibody can be bound to a solid support substrate, or conjugated with a detectable moiety, or both bound and conjugated (fluorescent moiety or enzyme moiety). (See Johnstone & Thorpe (1982), Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, for general considerations on the conjugation of). The binding of antibodies to solid support substrates is also well known in the art (for general discussion, see Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Publications, New York, England, and 92). (See Antibody Engineering-A Practical Guide, WH Freeman and Co.). Detectable moieties or labels contemplated with the present invention are fluorescent, metallic, such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14C, and iodination. Including, but not limited to, enzymatic and radioactive labels.

Purification of recombinant proteins For standard purification, see Marshak et al. (1996), “Stratesies for Protein Purification and Characterization. A laboratory course manual.” CSHL Press.

  The polynucleotide sequence of human ENDO180 mRNA is described in 5641 bases having an open reading frame (ORF) of 4439 bases (117 to 4441), accession number NM_006039, and the polypeptide sequence of 1479 amino acids (aa) has the gene identification number GI. : 110624474 with the accession number NP_006030. The mouse mRNA sequence is 5818 bases and is the accession number MMU56734 with an ORF of 1479aa.

  ENDO180 contains several protein domains as follows: 1-31aa SP (signal peptide); 41-161aa cysteine-enriched N-terminal domain; 180-228aa FNII (fibronectin type II) domain; 8 CDR (sugar recognition domain) ) Domain 1 CRD-8 CRD (235-360aa 1CRD, 382-505aa 2CRD, 521-645aa 3CRD, 669-809aa 4CRD, 825-951aa 5CRD, 972-1108aa 6CRD, 1161-1294aa 7CRD, 1261-1394aa14D) 1TM (transmembrane domain), 1437-1479aa cytoplasmic domain.

Reference to Sequence List The sequences described in this specification (SEQ ID NOs: 1-9) are text files created on March 23, 2010, titled “202-PCT1.ST25.txt” (file In size 36KB), which was filed with the present application via the USPTO's electronic filing system (EFS), which is hereby incorporated by reference in its entirety.
Example 1: Identification of ENDO180 overexpression by microarray hybridization test

  In accordance with the present invention, microarray hybridization techniques were used to find genes that are differentially regulated in diabetic nephropathy and renal fibrosis.

  The analysis of microarray-based gene expression was based on the analysis of human fibroblasts subjected to selected stimuli resulting in changes in extracellular collagen accumulation and proliferation characteristic of fibrosis. In accordance with the present invention, specific “fibrosis” DNA chips were first prepared, after which microarray hybridization experiments were performed with 19 different types of probes. The results were analyzed by a dedicated algorithm, and the inventors analyzed the selected gene set using physical informatics and scientific literature.

Example 2: Production of human anti-ENDO180 antibody The goal was to create an anti-ENDO180 antibody that binds to the extracellular portion of ENDO180 and internalizes the anti-ENDO180 antibody-cargo complex / conjugate. .

  Antigen production: Structural considerations in selecting an antigen for antibody production included a ligand-binding structure formed spatially by amino acids 1-522 (SEQ ID NO: 9).

  Recombinant ENDO180 antigen was produced by cloning nucleotides 1-1566 of the human ENDO180 polynucleotide into an expression vector containing a FLAG epitope. The polynucleotide sequence of the recombinant clone is set forth in SEQ ID NO: 3, and the amino acid sequence is set forth in SEQ ID NO: 4. The vector was transfected into 293T cells and a clone expressing a partial extracellular domain (59 KD) of human ENDO180 was identified. ECDhENDO180-FLAG protein was isolated as follows: Approximately 2.2 liters of conditioned medium was filtered through a 0.22 um filter. Medium was loaded on M2 agarose (5 ml, Sigma) previously equilibrated (with TBS) at a flow rate of 1 ml / min. The resin was washed with 10 column volumes using TBS, then 10 volumes with 50 mM Tris (pH 7.5), 1 M NaCl, and finally with 10 volumes of TBS. Elution was performed with 10 ml of 0.5 mg / ml FLAG peptide in TBS, pH 7.5 (final pH). The resin was incubated with elution buffer for 20 minutes before starting column efflux. The sample was concentrated and the FLAG peptide was removed using VivaSpin ™ (cut-off 10 Kd). Glycerol was added to a final 10% and the protein was snap frozen in liquid nitrogen.

  Identification of miniantibodies: Di Niro et al, 2007. Construction of miniatures for the in vivo study of human autoimmune diseases in animal models. Miniantibodies were identified according to the method disclosed in BMC Biotechnology 7:46.

  Certain preferred antibodies according to the present invention are recombinant polypeptides comprising heavy and light chain CDR3 domains having the amino acid sequences set forth in SEQ ID NO: 7 and SEQ ID NO: 8.

Example 3: Anti-ENDO180 monoclonal antibody Methods and overview The mAb and MB were labeled with CypHer5e according to the manufacturer's instructions (GE Healthcare).
2. Labeled / unlabeled mAb and MB were tested for binding to purified ENDO180 extracellular domain by standard ELISA.
Clones 6D6, 8D8, 8E7, and 9G10 still showed saturation binding to ENDO180DCTLD3-8-FLAG after labeling with CypHer5E. In contrast, the binding of clone 8D2 and MB (miniantibody) was significantly impaired by labeling.
3. Internalization assays were performed according to methods for those skilled in the art.
Cells expressing ENDO180 incubated with labeled mAbs 6D6, 8D8, 8E7, and G10 showed some increase in fluorescence after 1 hour at 37 ° C. Most notably, the same cells incubated with labeled MB showed strong fluorescence. This increase in fluorescence was not seen at 40C in control cells or ENDO180 cells. The control mAb (10F12) had no effect.
4). Kinetic experiments Based on the results of the previous experiment, mAbs 6D6, 8D8 and MB were tested. In kinetic experiments from 10 minutes to 1 hour, mAbs 8D8 and MB showed the best internalization. All negative controls, control cells (4 ° C., indicated as 40C in some figures), and control mAbs were negative.

Experimental details and results An anti-ENDO180 monoclonal antibody (mAb) against the most N-terminal domain (1-522aa) of human ENDO180 (SEQ ID NO: 9) was generated and screened for internalization in an essentially ENDO180-specific manner; Conjugated to the fluorophore CypHer5E.

  mAb production: Approximately 8 liters of each hybridoma is grown in DMEM medium supplemented with 5% FBS IgG FREE, 1% Penstrep, 1% L-glutamine, 50 μg / ml gentamicin, 2.5 μg / ml amphotericin B (fungizone). I let you. About 60 ml was obtained from the cell line flask at an antibody concentration of about 200 μg / ml. The growth period was one month. Purification was performed using a protein A sepharose column followed by a two cycle size separation column.

  mAbs 8D8 (E3-8D8), 6D6, 8E7, 9G10, 8D2 were selected for conjugation to the target moiety.

  The mAb was covalently coupled to the red-excitable, pH-sensitive fluorescent cyanine dye CypHer5E (Catalog Number pA15405, Amersham) at a 20: 1 molar ratio of CypHer5E: antibody according to the manufacturer's instructions. The fluorophore was excited at acidic pH as seen in the endosome. Thus, these antibodies that are internalized appear as fluorescent signals in intracellular vesicles. The mAb was covalently attached to biotin using EZ-Sulfo-NHS-biotin (Pierce, catalog number 21217, lot number CE49927). Biotinylation was performed for 30 minutes at room temperature using biotin: antibody in a molar ratio of 20: 1 according to manufacturer's instructions. After a further 2 hours at room temperature against fresh PBS solution, the antibody solution was dialyzed overnight at 4 ° C. against PBS solution after covalently binding the mAb to CypHer5E or biotin. Labeling of mAbs and scFvs (SEQ ID NO: 6, also referred to as “miniantibodies”) was performed using CypHer5E according to the manufacturer's instructions (GE Healthcare).

  In the absence of collagen, internalization of the conjugated receptor was performed (ligand independent internalization). The following clones were found to secrete antibodies that specifically bind ENDO180: clones 6D6, 8D8, 8E7, and 9G10 were labeled against ENDOL80DCTLD3-8-FLAG itself and with CypHer5E. Saturated bond was shown. Clone 8D2 showed limited uptake into cells after labeling, while clone 10F12 showed significant saturation binding to ENDO180DCTLD3-8-FLAG, but it decreased after labeling.

  Cells expressing ENDO180 (NRK52E-ENDO180) and control cells (NRK52E) were incubated at 37 ° C. with the indicated anti-ENDO180 mAb or control mAb covalently linked to CypHer5E. The mAb was also incubated with ENDO180 cells at 4 ° C. Cells were seeded in 96-well plates and fluorescence was measured by Biosystems Analyst AD and HT (excitation 530 nm, emission 590 nm, dichroism 560 nm). There was a steady increase in fluorescence of ENDO180 cells with one mAb (E3-8D8) at 37 ° C. In contrast, at 4 ° C., no fluorescence was seen in control cells or control mAbs. In NRK52 cells expressing ENDO180, antibody binding and internalization were tested at a permissive temperature (37 ° C.) and a non-permissive temperature (4 ° C.), and after 1 hour. FIG. 2A shows the fluorescence levels of cells expressing ENDO180 at permissive temperature (37 ° C.) and non-permissive temperature (4 ° C.). Clones 6D6 and 8D8 and scFv (SEQ ID NO: 6) show the highest level of internalization. FIG. 2B shows that 8D8 showed non-specific binding.

  The ENDO180 receptor has been shown to be an internalization and recycling receptor (Howard and Isacke, 2002, JBC 277, 35: 32320-31), all antibodies produced were internalized at the same rate or amount. It is not done. mAbs 8D8 and G7V scFv showed unexpected internalization. mAb 10F2 was still not internalized after 8 hours.

  We investigated the dynamics of internalization. NRK52 cells stably expressing human ENDO180-FLAG or empty vector were seeded in TC grade black 96 well plates at a density of 6000 cells / well. After 24 or 48 hours, mAb (5 ug / ml in growth medium) was added to the wells at 100 ul / well, either at room temperature or on ice (for control plates). The plate was rapidly incubated several times at 37 ° C. Control plates were kept on ice for the required time. At each time point, the plate was washed 3 times in 200 ul of cold PBS. After the final wash, 100 ul of cold PBS was added and the plate was read using Analyst at Ex 610 nm / Em 670 nm.

  2C-2E show the time course of 10 minutes to 1 hour of internalization of anti-ENDO180-CypHer5E conjugate by cells expressing ENDO180. FIG. 2C shows internalization of 8D8 labeled with CypHer5E. FIG. 2D shows the internalization of scFv G7V (SEQ ID NO: 6) labeled with CypHer5E. FIG. 2E shows the internalization of 10F2 labeled with CypHer5E.

  Figures 2F-2H show the time course of 1-8 hours of internalization of anti-ENDO180-CypHer5E conjugate by cells expressing ENDO180. FIG. 2F shows internalization of 8D8 labeled with CypHer5E. FIG. 2G shows internalization of scFv G7V (SEQ ID NO: 6) labeled with CypHer5E. FIG. 2E shows the internalization of 10F2 labeled with CypHer5E.

  The hybridoma cell line E3-8D8 (also referred to as 8D8), which secretes the monoclonal antibody E3-8D8, was obtained from Ghent University, Department of Biomedical Molecular Co-ordinary Colors of Ghent University, Department of Biomedical Molecular Co-ordinary Colors. The deposit number was LMBP 7203 CB. The deposit was made on March 9, 2010, tested on March 18, 2010 and shown to be alive.

  A composition for systemic uptake of a drug across a mucosa comprising a polyethylene glycol-chitosan conjugate, the polyethylene glycol-chitosan conjugate comprising a chitosan or chitosan derivative moiety, a polyethylene glycol or polyethylene glycol derivative moiety, The composition is formulated for delivery to the mucosa.

A general method for preparing substrate-drug conjugates according to the present invention involves covalently attaching at least one therapeutic or diagnostic agent to a substrate. Certain cytotoxic agents useful for anticancer therapy are relatively insoluble in serum. Some are also very toxic in unconjugated forms, and their toxicity is greatly reduced by conversion to conjugates. By converting a relatively poorly soluble drug into a more soluble conjugate, such as glucuronide, the solubility in the serum aqueous phase and through the cell walls of veins, arteries, or capillaries, the tumor The ability to reach interstitial fluid while soaking is improved. In fact, converting certain toxic substances such as aromatic or cycloaliphatic alcohols, thiols, phenols, and amines into liver glucuronides detoxifies them and excretes them in the urine more easily. Is the method.
Example 4: Internalization of antibody conjugates in vitro and in vivo

  FIG. 3 shows the internalization of biotin by the anti-ENDO180 mAb into mouse unilateral ureteral obstructed kidney.

  The following experiment was designed to assay ENDO180 antibody accumulation in tissues expressing ENDO180. Unilateral ureteral obstruction (UUO) was performed in mice. On day 7 of UUO surgery, kidney ENDO180 levels were higher than contralateral kidney (data not shown).

  Seven days after UUO surgery, mice were injected with 3 mg / kg E3-8D8-biotin conjugate or NMIgG-biotin conjugate, and 24 hours later, kidney. The uptake levels of E3-8D8-biotin conjugate and NMIgG-biotin (normal mouse IgG control) conjugate in unilateral ureteral obstruction (UUO) kidney and contralateral kidney were examined by Western blot (WB). The same amount of kidney total protein extract was examined by WB using goat anti-biotin HRP (Cell Signaling, # 7075).

  FIG. 4 shows internalization of anti-ENDO180-CypHer5E conjugate into myelomonocytic human leukemia MonoMac cell line expressing ENDO180. MonoMac cells were incubated with E3 8D8-CypHer5E or E3 8D2-CypHer5E. Cells were washed twice with cold PBS and internalization was measured by FACS using FL-1 or FL-4 filters. E3 8D8 bound to ENDO 180 with higher affinity than E3-8D2 (data not shown). 8D2 is a mAb that binds to ENDO180 with a lower affinity than 8D8.

Example 5: Linkage of antibody to therapeutic agent a. MB: Complete human protamine (approximately 50a.a) is cloned directly downstream of the heavy chain constant region. A similar strategy was used using an anti-HIV-1 gp120 recombinant Fab fragment with a bicistronic vector expressing VH-CH1-protamine from one promoter and VKCK light chain from another promoter (Chen et al. Gene Therapy (1995) 2, 116-123). This construct was found to have in vivo anti-tumor activity (Song et al., Nature Biotech. 2005.23 (6), pg. 709-717). In another study, protamine was fused downstream of a single chain antibody against ErbB (Li et al., Cancer Gene Therapy 8 (8), 2001, pg. 555-565).
b. The mAb: CDR domain of monoclonal antibody 8D8 was sequenced and cloned in a manner similar to MB to allow fusion with it and genetic manipulation of protamine.
c. To link an antibody or antigen-binding fragment thereof (MB, isolated mAb, scFv, F'ab, etc.) to a therapeutic agent using one or more of a peptide, nucleic acid, chemical linker or lipid linker . Use standard methods.

Example 6: In situ hybridization in cancer tissue samples Using in situ hybridization techniques, samples of human tissue from cancer patients were tested for the expression of ENDO180. Tissue samples were analyzed by a skilled pathologist. The results showed the following expression pattern.
1. Renal cell carcinoma (RCC)
In two different renal cell carcinoma types, clear cell carcinoma (4 cases) and papillary carcinoma (1 case), high levels of ENDO180 mRNA expression were seen in all five sarcoma-like regions tested. Sarcomatoid cancers develop in all major types of renal cell carcinoma (clear cell carcinoma, papillary carcinoma, chromophoric carcinoma, and collecting duct carcinoma). They are found in about 1-1.5% of all adult renal tumors with an aggressive clinical course and poor prognosis.
Endo180 mRNA expression is also found in stromal cells, non-neoplastic stromal cells, and glomerular cells within tumor tissue, with some sample-to-sample variation in cell volume and signal intensity.
2. Ovarian cancer:
In most borderline serous papillary carcinomas (7/8) presented in this study, ENDO180 expression was seen in a subset of tumor cells at various intensities.
Expression of ENDO180 mRNA in ovarian cancer cells is seen in 8 of 21 cases with some sample-to-sample variation in both the amount of cells expressed and the signal intensity.
A high intensity signal of ENDO180 mRNA expression was found in a subset of stromal cells around the tumor and a subset of ovarian stromal cells (94% and 100%). The expression of ENDO180 mRNA was also detected in normal epithelial single cells.
3. Transitional cell carcinoma (TCC):
Endo180 expression was weak in 4 cases of primary transitional cell (urinary tract) carcinoma (18/27 cases) and metastatic tumors (of lymph nodes) of most bladder presented in this study . A high intensity signal of ENDO180 mRNA expression was seen in a subset of stromal cells surrounding the tumor.
4). breast cancer:
Endo180 mRNA expression was found around most tumors in invasive cancer cases (84%). There was no constant expression pattern in epithelial cells.

Example 7: Animal model for testing compounds in the treatment of fibrosis Exemplary molecules and conjugates of the invention are tested for efficacy in the treatment of subjects suffering from fibrosis and fibrotic diseases. The following animal model is presented as a non-limiting example for use in practice. Other animal models are also considered.

  A useful way to assess the development of kidney disease with fibrosis and glomerulosclerosis is to characterize gene expression in an established animal model of kidney disease. Examples of such models are: (i) fa / fa rats: animals with genetic defects in the leptin receptor that develop insulin-resistant diabetes (type 2 diabetes) with progressive diabetic nephropathy, And (ii) GK rats: including but not limited to genetically engineered NIDDM phenotype rats. Another animal model in which mainly renal fibrosis is evident but no background of diabetes exists is unilateral ureteral obstruction (UUO), where interstitial fibrosis occurs rapidly within days after obstruction. 5/6 nephrectomy is another useful animal model for chronic renal failure (CRI) where fibrosis is evident.

  A further aspect of the study may be based on an in vitro model system involving culturing human fibroblasts in vitro under conditions that mimic various parameters of the cellular microenvironment present in CRI and fibrosis. These include high concentrations of glucose (modeling of hyperglycemia), low concentrations of glucose, hypoxia (both modeling of ischemic conditions occurring in the kidney after fibrosis and glomerulosclerosis), and fibrosis Treatment with TGF-b, one of the pathogenic factors recognized in the disease. Such in vitro model systems can complement animal models in several important aspects. First, the system is specific for fibroblasts and thus there is no interference that is often found in complex tissues containing many cell types. Second, unlike animal models, cells are of human origin. Furthermore, because the disorder is specific and at various concentrations and durations, it is possible to investigate both acute and chronic responses.

Example 8: Animal model for testing compounds in cancer therapy The effectiveness of the exemplary molecules and conjugates of the present invention in the treatment of subjects suffering from cancer and other proliferative and metastatic diseases. The following animal model is presented as a non-limiting example for use in testing for: Other animal models are also considered.
Transplantation in immunodeficient mice

  NOD / SCID mice are defective in both lymphatic and bone marrow function and readily tolerate long-term survival of human hematopoietic cells. The transplantation of human bone marrow from NOD / SCID mice to human / mouse chimeras is well documented.

Bertolini et al (2000. Blood 96-282) used NOD / SCID mice as an advanced non-Hodgkin lymphoma model. A Namalva cell line derived from Epstein-Barr virus positive Burkitt non-Hodgkin lymphoma was used. Cells (10 × 10 ⁶ ) were injected intraperitoneally into 6-8 week old mice. An intraperitoneal tumor that can be measured with calipers was formed at the injection site. The formula “width 2 × length × 0.52” was applied to estimate the volume of the spheroid (see Bohem et al (1997) for further reference).

  The model used in the following studies is based on transgenic SCID mice expressing human GM-CSF. This cytokine expression allowed a good transplantation of the relevant cell line in SCID mice. Miyakawa et al (1996) details the production of hGM-CSF SCID transgenic mice. Fukuchi et al (1998) show that retinoic acid resistant leukemia can be established in these transgenic mice. The model consists of UF-1 cells, an RA-resistant APL cell line established in the laboratory, and is transplanted into these transgenic SCID mice, causing the appearance of subcutaneous tumors. Kinjo et al (2000) uses this model to test arsenite, a specific therapeutic agent used in cases of RA-resistant acute promyelocytic leukemia (APL).

  Lewis et al (1998) is a mouse strain NOD that is more prominently immunodeficient, with functional defects in both T and B cells and significant impairment in macrophage, natural killer cell, and hemolytic complement activity. / SCID was used. These mice form a good model for this disease because they can be transplanted with cells from cancer patients, resulting in a relatively high success rate.

Claims (18)

An anti-ENDO180 antibody,
The anti-ENDO180 antibody is
a. An isolated monoclonal antibody produced by the hybridoma cell line E3-8D8 deposited with BCCM as accession number LMBP 7203CB;
b. A humanized antibody of (a), and c. (A) selected from the group consisting of chimeric antibodies,
An anti-ENDO180 antibody, wherein the antibody is internalized into the cell when contacted with a cell expressing ENDO180 . A composition comprising the anti-ENDO180 antibody of claim 1 and a pharmaceutically acceptable carrier.   The composition of claim 2, further comprising a moiety selected from the group consisting of a detectable label, a cytotoxic agent, and a therapeutic agent. a) an antibody according to claim 1,
b) a moiety selected from the group consisting of a detectable label, a cytotoxic agent, and a therapeutic agent;
c) a conjugate, optionally comprising a) linking a) to b).   The conjugate according to claim 4, wherein the linker is selected from a polypeptide linker, a peptide linker, a lipid linker, or a nucleic acid linker.   The moiety is a therapeutic agent, and the therapeutic agent is from an antisense compound, a chemically modified siRNA compound, an unmodified siRNA compound, a chemically modified shRNA compound, an unmodified shRNA compound, a chemically modified miRNA compound, and an unmodified miRNA compound. 5. The composition of claim 3 or the conjugate of claim 4, wherein the composition is an inhibitory oligonucleotide selected from the group consisting of:   7. The composition or conjugate of claim 6, wherein the inhibitory oligonucleotide is a chemically modified siRNA compound.   A composition comprising the conjugate according to any one of claims 4 to 7 and a pharmaceutically acceptable carrier.   4. The composition of claim 3, wherein the carrier comprises lipid particles, polysaccharide particles, liposomes or combinations thereof.   4. The composition of claim 3, wherein the carrier comprises lipidated polysaccharide particles.   11. The composition of claim 10, wherein the lipidated polysaccharide particles comprise a lipidated glycosaminoglycan. The composition according to claim 9 or 10, wherein the anti-ENDO180 antibody is immobilized on the particles or liposomes.   13. A composition according to claim 9, 10 or 12, wherein the portion is encapsulated within the particle or liposome.   14. A composition according to any one of claims 3 and 8-13 for the treatment of a disease selected from the group consisting of cancer and fibrosis.   Hybridoma cell line E3-8D8 deposited with BCCM under accession number LMBP 7203CB. The antibody is attached to the surface of the delivery particle, said delivery particles, detectable labels, include at least one portion selected from the group consisting of cytotoxic agents, and therapeutic agents, to claim 1 The described anti-ENDO180 antibody .   5. The conjugate of claim 4, wherein the conjugate is for use in a method of delivering the moiety to cells expressing ENDO180 in a subject by administering the conjugate.   14. The composition of claim 13, wherein the composition is for use in a method of delivering the moiety to cells expressing ENDO180 in a subject by administering the composition.